Laboratory techniques in rabies
Fifth edition Volume 1
Edited by
Charles E. Rupprecht LYSSA LLC Atlanta, Georgia, USA
Anthony R. Fooks Animal and Plant Health Agency Addlestone, Surrey, United Kingdom
Bernadette Abela-Ridder Department of Control of Neglected Tropical Diseases World Health Organization Geneva, Switzerland Laboratory techniques in rabies Fifth edition Volume 1
Edited by
Charles E. Rupprecht LYSSA LLC Atlanta, Georgia, USA
Anthony R. Fooks Animal and Plant Health Agency Addlestone, Surrey, United Kingdom
Bernadette Abela-Ridder Department of Control of Neglected Tropical Diseases World Health Organization Geneva, Switzerland /DERUDWRU\WHFKQLTXHVLQUDELHVĺIWKHGLWLRQ9ROXPH&KDUOHV(5XSSUHFKW$QWKRQ\5)RRNV%HUQDGHWWH$EHOD5LG GHUHGLWRUV
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)RUHZRUG iv Preface v List of abbreviations and acronyms used in this manual vi
Part 1. General considerations &KDSWHU ,QWURGXFWLRQ &KDSWHU /\VVDYLUXVHV &KDSWHU %LRVDIHW\ &KDSWHU 7KHUROHRIGLDJQRVWLFVLQVXUYHLOODQFH &KDSWHU $QRYHUYLHZRIDQWHPRUWHPDQGSRVWPRUWHPWHVWVIRUGLDJQRVLVRI human rabies &KDSWHU +LVWRSDWKRORJLFDOWHFKQLTXHVLQWKHODERUDWRU\GLDJQRVLVRIKXPDQUDELHV &KDSWHU %UDLQUHPRYDO
Part 2. Detection of virus &KDSWHU 9LUXVLVRODWLRQLQDQLPDOV WKHPRXVHLQRFXODWLRQWHVW &KDSWHU 9LUXVLVRODWLRQLQFHOOFXOWXUH WKHUDELHVWLVVXHFXOWXUHLQIHFWLRQWHVW &KDSWHU 7UDQVPLVVLRQHOHFWURQPLFURVFRS\LQUDELHVGLDJQRVLV ultrastructural studies and research
Part 3. Demonstration of antigens &KDSWHU 7KHGLUHFWĻXRUHVFHQWDQWLERG\WHVW &KDSWHU 7KHGLUHFWUDSLGLPPXQRKLVWRFKHPLVWU\WHVWIRUWKHGHWHFWLRQ of lyssavirus antigens &KDSWHU ,PPXQRKLVWRFKHPLVWU\ &KDSWHU $QWLJHQLFW\SLQJRIO\VVDYLUXVHVE\PRQRFORQDODQWLERGLHV &KDSWHU 8VHRIDUDSLGVNLQELRSV\WHFKQLTXHIRUKXPDQUDELHV antemortem diagnosis &KDSWHU 'HPRQVWUDWLRQRIO\VVDYLUXVDQWLJHQVE\ĻRZF\WRPHWU\ &KDSWHU 5DSLGLPPXQRFKURPDWRJUDSKLFWHVWVIRUWKHGHWHFWLRQRI rabies virus antigens in brain material &KDSWHU 0DVVVSHFWURPHWU\EDVHGSURWHRPLFDSSURDFKHVIRU the detection of rabies virus peptides
Part 4. Demonstration of viral antibodies &KDSWHU 7KHUDSLGĻXRUHVFHQWIRFXVLQKLELWLRQWHVW &KDSWHU 7KHĻXRUHVFHQWDQWLERG\YLUXVQHXWUDOL]DWLRQWHVW &KDSWHU $QLQGLUHFWĻXRUHVFHQWDQWLERG\WHVWIRUWKHGHWHFWLRQ RIUDELHVYLUXVLPPXQRJOREXOLQ*DQGLPPXQRJOREXOLQ0DQWLERGLHV &KDSWHU 7KHPRXVHQHXWUDOL]DWLRQWHVW &KDSWHU 'HPRQVWUDWLRQRIO\VVDYLUXVDQWLERGLHVE\SVHXGRW\SH YLUXVPLFURQHXWUDOL]DWLRQDVVD\V &KDSWHU $VLPSOLĺHGĻXRUHVFHQFHLQKLELWLRQPLFURWHVWIRUWKH determination of rabies virus neutralizing antibodies &KDSWHU 7KHLPPXQRSHUR[LGDVHLQKLELWLRQDVVD\ &KDSWHU 'HPRQVWUDWLRQRIUDELHVYLUXVDQWLERGLHVE\WKHFRXQWHULPPXQRHOHFWURSKRUHVLVWHVW
Appendix 1 List of contributors Appendix 2 WHO collaborating centres on rabies, neurovirology, viral zoonoses and zoonoses control Laboratory techniques in rabies Fifth edition
Laboratory techniques in rabies Foreword
)RUPRUHWKDQ\HDUVKXPDQVKDYHOLYHGLQIHDURIDELWHIURPDUDELG DQLPDOVRPXFKVRWKDWWKHĺUVWZULWWHQDFFRXQWRIUDELHVLQWKHUGFHQWXU\}%& VHWWKHSHQDOW\IRUDQRZQHUpVGRJELWLQJDQRWKHULQGLYLGXDODWqWZRWKLUGVRID PLQHRIVLOYHUrRUDERXWDKDOIGD\pVZRUN7RGD\RXUIRFXVLVPRUHRQSUHYHQWLQJ rabies and advocating for its elimination, rather than imposing penalties, and our XQGHUVWDQGLQJRIWKHYLUXVKDVJUHDWO\LPSURYHGVLQFHWKHUGFHQWXU\%& 7KH)RRGDQG$JULFXOWXUH2UJDQL]DWLRQRIWKH8QLWHG1DWLRQV )$2 WKH:RUOG Organisation for Animal Health (OIE) and the World Health Organization (WHO) KDYHSULRULWL]HGDFWLRQDJDLQVWUDELHVDQGWRJHWKHUZLWK0HPEHUFRXQWULHVKDYH VHWDJRDORI]HURUDELHVGHDWKVE\'LDJQRVWLFVDUHFUXFLDOLQDWWDLQLQJWKLV JRDO New laboratory techniques and advancements in science have yielded better GLDJQRVWLFWHFKQLTXHVDQGFRQWUROVWUDWHJLHVWRDLGWKHPRUHWKDQELOOLRQSHRSOH PDLQO\ FKLOGUHQ LQ $VLD DQG $IULFD ZKR DUH WKUHDWHQHG E\ WKH YLUXV HYHU\ GD\ 5DELHV LV D SUHYHQWDEOH GLVHDVH \HW GHVSLWH WKH DYDLODELOLW\ RI HIĺFDFLRXV DQG DIIRUGDEOHYDFFLQHVPRUHWKDQSHRSOHZRUOGZLGHGLHDJRQL]LQJGHDWKV HYHU\\HDUIURPWKHGLVHDVH No diagnostic tests are available to detect the rabies virus before the onset of FOLQLFDOGLVHDVHDQGIXUWKHUUHVHDUFKRQGLDJQRVWLFWHFKQLTXHVLQWKHĺHOGRIUDELHV LVWKHUHIRUHSDUDPRXQW7KHLPSDFWRIVXLWDEOHODERUDWRU\FDSDFLW\RQVXUYHLOODQFH DQGHOLPLQDWLRQRIWKHGLVHDVHZRUOGZLGHLVHYLGHQW 7KH 2,(pV 0DQXDO RI GLDJQRVWLF WHVWV DQG YDFFLQHV IRU WHUUHVWULDO DQLPDOV provides internationally agreed standards for the production and control of vali GDWHGYHWHULQDU\GLDJQRVWLFPHWKRGVDQGYDFFLQHVIRUXVHLQDQLPDOV7KHIRXUWK HGLWLRQRI:+2pV/DERUDWRU\WHFKQLTXHVLQUDELHVKDVEHHQDJXLGLQJUHIHUHQFH IRUPDQ\UDELHVODERUDWRULHV7KHĺUVWHGLWLRQ VWDWHGWKDWqUDELHVUHVHDUFK LVIDUIURPVWDWLFrDQGVLQFHLWVSXEOLFDWLRQPRUHWKDQ\HDUVDJR2,(DQG:+2 KDYHZRUNHGWRHYDOXDWHVXEVHTXHQWDGYDQFHPHQWVLQODERUDWRU\WHFKQLTXHVLQ UDELHV7KLVĺIWKHGLWLRQSURYLGHVLQVLJKWLQWRYDOLGDWHGPHWKRGVUHFRPPHQGHGIRU XVHLQGLDJQRVWLFODERUDWRULHVEXWLWDOVRLQFOXGHVUHVHDUFK:KLOHQRWFXUUHQWO\ applicable to all settings, these research methods may stimulate the development RILPSURYHGWHFKQLTXHVIRUGLDJQRVLVRIUDELHVLQWKHIXWXUH,PSURYHGGLDJQRVWLFV will strengthen surveillance of the disease, leading to enhanced control of rabies ZKHUHLWLVPRVWQHHGHG
iv Laboratory techniques in rabies Fifth edition
Laboratory techniques in rabies Preface
5DELHVKDVDQHQRUPRXVLPSDFWRQERWKDJULFXOWXUHDQGFRQVHUYDWLRQELRORJ\EXW LWVJUHDWHVWEXUGHQLVXQGHQLDEO\RQSXEOLFKHDOWK$VVXFKURXWLQHPHWKRGVIRUUDSLG ULVNDVVHVVPHQWDIWHUKXPDQH[SRVXUHVWRUDELHVDVZHOODVDSSOLFDWLRQVIRUODERUD WRU\EDVHGVXUYHLOODQFHSURGXFWLRQRIELRORJLFDOVDQGPDQDJHPHQWRIWKLVLQIHFWLRXV GLVHDVHDUHFULWLFDO*LYHQLWVPDQGDWHWRLPSURYHKXPDQKHDOWKDQGFRQWUROGLVHDVH DPRQJLWV0HPEHU6WDWHV:+2KDVOHGWKHSURGXFWLRQRIWKLVĺIWKHGLWLRQRILaboratory techniques in rabies
'XULQJWKHPRUHWKDQ\HDUVWKDWKDYHHODSVHGVLQFHWKHĺUVWHGLWLRQZDVSXEOLVKHG methods of viral diagnosis, characterization of pathogens and production of biologicals KDYHDGYDQFHG$WWKDWWLPHRQO\DVLQJOHHWLRORJLFDODJHQWZDVUHFRJQL]HGDVFDXVLQJ UDELHV'HWHFWLRQRI1HJULERGLHVZDVWKHVWDQGDUGIRUGLDJQRVLV1HUYHWLVVXHEDVHG YDFFLQHVZHUHWKHQRUP&RPELQDWLRQXVHRIYDFFLQHVDQGUDELHVLPPXQRJOREXOLQVLQ KXPDQSURSK\OD[LVZDVQRWVWDQGDUG*OREDOHOLPLQDWLRQRIFDQLQHUDELHVZDVPHUHO\D GUHDP5DELHVLQZLOGOLIHZDVPDQDJHGYLDSRSXODWLRQUHGXFWLRQ$OORIWKDWKDVFKDQJHG IRUWKHEHWWHU
In the ensuing decades, further advancements in detection, prevention and control of lyssaviruses have been monitored by regular meetings of WHO experts, international UHVHDUFKJURXSVDQGFRXQWULHVLQZKLFKUDELHVLVHQGHPLF7KHVHFRQGHGLWLRQRIWKH PDQXDOZDVSXEOLVKHGLQ WKHWKLUGLQ DQGWKHIRXUWKLQ 7KHODWH0DUWLQ .DSODQDQG+LODU\.RSURZVNLZHUHLQVWUXPHQWDOLQHGLWLQJWKHSUHYLRXVHGLWLRQVDVZDV LQSXW RQ WKH IRXUWK HGLWLRQ E\ )UDQÄRLV;DYLHU 0HVOLQ QRZ UHWLUHG IURP :+2 ,QLWLDO SODQVIRUSUHSDUDWLRQRIWKLVHGLWLRQZHUHPDGHLQDQGLWVFRQWHQWVZHUHGLVFXVVHG DWWKH:+2([SHUWPHHWLQJRQUDELHV %DQJNRN7KDLODQG DQGPRGLĺHGLQUHVSRQVH
7KLVĺIWKHGLWLRQRILaboratory techniques in rabiesFRQWDLQVGHWDLOHGFKDSWHUV ZULWWHQE\PRUHWKDQDXWKRUVIURP$IULFDWKH$PHULFDVDQG(XUDVLD7KHWH[WZDV SHHU UHYLHZHG E\ 'U 0DWWKLDV 6FKQHOO +HDG RI WKH :+2 &ROODERUDWLQJ &HQWUH IRU Neurovirology; Professor Thiravat Hemachudha, Head of the WHO Collaborating Center IRU5HVHDUFKDQG7UDLQLQJRQ9LUDO=RRQRVHV DQG'U$VHID'HUHVVD7HDP/HDGHURI =RRQRVHV5HVHDUFKDWWKH(WKLRSLDQ3XEOLF+HDOWK,QVWLWXWH7KH}PDQXDOIRFXVHVRQ the basic methods for detection of lyssavirus antigens, antibodies and nucleic acids and the relevance of their use under different operating conditions, from the basic to WKHDGYDQFHG7KHFKDSWHUVRQROGHUOHVVVHQVLWLYHWHFKQLTXHVXVHGWRGHWHFW1HJUL bodies have been removed, as have those chapters on methods of vaccine production given the progress made in the commercial use of tissue culture products in human and YHWHULQDU\PHGLFLQH5HFRPPHQGDWLRQVIRUWKHSUHSDUDWLRQVRIDQWLERGLHVE\KRPROR gous or heterologous production have been replaced by newer methods in an effort to promote a next generation of less expensive and more readily available immunoglobu OLQVLQWKHIXWXUH2WKHUEDVLFFKDSWHUVKDYHEHHQUHWDLQHGDQGXSGDWHGDQGPRUHWKDQD GR]HQDGGHG(DFKRIWKHSURWRFROVGHVFULEHGDUHSUHVFULSWLYHDQGVKRXOGEHIROORZHG SRLQWE\SRLQWLQWKHODERUDWRU\
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v Laboratory techniques in rabies Fifth edition
List of abbreviations and acronyms used in this manual
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vi Abbreviations Laboratory techniques in rabies Fifth edition
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xii Abbreviations Laboratory techniques in rabies Fifth edition
Part 1. General considerations
xiii Laboratory techniques in rabies Fifth edition Part 1. General considerations
Chapter 1 Introduction
$VLPSOLĺHGHDUO\WKFHQWXU\YLHZRIWKHKLVWRULFDOUDELHVODERUDWRU\PD\KDYH been predisposed towards a linear, passive role of sample receipt and diagnosis RQO\7RGD\WKHLGHDOUROHRIWKHPRGHUQUDELHVODERUDWRU\LVPRUHFRPSOH[DQG PXOWLGLPHQVLRQDO 7DEOH :KLOHVRPHODERUDWRULHVPD\SHUIRUPRQHRURQO\ a few core functions, others conduct much more diverse activities, especially the reference laboratories of the World Organisation for Animal Health (OIE) and the :RUOG +HDOWK 2UJDQL]DWLRQ :+2 0RUHRYHU EHFDXVH UDELHV LV D IXQGDPHQWDO disease of nature, the laboratory should be engaged not only in diagnostics per se but also in actively maintaining a central role in the assessment, planning and investigation of cases of rabies in humans, domestic animals and wildlife locally and abroad as part of routine prevention and control using a One Health approach )LJ 7KLVFULWLFDOUHVSRQVLELOLW\IRULQYROYHPHQWLQKXPDQYHWHULQDU\DQGHQYL URQPHQWDOKHDOWKKDVJXLGHGWKHSURGXFWLRQRIWKLVĺIWKHGLWLRQRILaboratory tech- niques in rabies and informed the practical approach requested by countries in which canine rabies is endemic to support the goal of eliminating human deaths IURPGRJPHGLDWHGUDELHVE\
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Introduction Laboratory techniques in rabies Fifth edition Part 1. General considerations
Incident Investigation Sample collection
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At a minimum, all rabies laboratories should be capacitated to perform diagnosis XVLQJ WKH qJROG VWDQGDUGr GLUHFW ĻXRUHVFHQW DQWLERG\ WHVW ')$7 7KLV} KLJKO\ VHQVLWLYHDQGVSHFLĺFPHWKRGKDVEHHQLQURXWLQHXVHIRUPRUHWKDQ\HDUV,Q retrospect, this manual is a culmination not only of reliable laboratory techniques that have stood the test of time but also of newer protocols for detection of viral antigens, antibodies and amplicons, production of relevant biologicals, where DSSOLFDEOHDQGDSSOLHGUHVHDUFK The current design relies on the successful outline of previous editions, starting ZLWKJHQHUDOFRQVLGHUDWLRQVDERXWWKHGLVHDVH7KLVĺIWKHGLWLRQLVVHSDUDWHGLQWR WZRYROXPHVWRUHĻHFWRSHUDWLRQDODQGUHVHDUFKIRFXVHGFRQWHQWDQGLVDYDLODEOH online only to facilitate rapid electronic updates as further advances in laboratory WHFKQLTXHVEHFRPHDYDLODEOH$VUDELHVLVFDXVHGE\QXPHURXVO\VVDYLUXVHVDQ introduction (Chapter 2) to the genus is an appropriate reminder that rabies virus is only one viral species of special concern and to promote further pathogen disco YHU\IURPDJOREDOSHUVSHFWLYH%LRVDIHW\ Chapter 3) is of paramount importance WRDOOODERUDWRULDQVDQGWKHUHIRUHZDUUDQWVDVHSDUDWHFKDSWHU1RWDEO\KXPDQDQG DQLPDOGLDJQRVWLFVDOLNHPXVWRSHUDWHZLWKLQDORJLFDOVSKHUHIRUODERUDWRU\EDVHG surveillance to operate (Chapter 4 +XPDQUDELHVLVXQGHUUHSRUWHGRQDOOFRQWL QHQWVDQGWKHFXUUHQWJXLGDQFHVHHNVWRDGGUHVVWKLVVKRUWFRPLQJ Chapters 5 and 6 &OHDUO\DEDVHVXVSLFLRQRIKXPDQHQFHSKDOLWLVLVQRWVWUDLJKWIRUZDUGDQG VKRXOGEHDSSURDFKHGZLWKLQDVWUXFWXUHGIUDPHZRUNRIGLIIHUHQWLDOGLDJQRVHV(1) Advanced neuroimaging may be useful to consider an infectious etiology, inclu ding rabies (2)$QWHPRUWHPGHWHFWLRQRIVSHFLĺFLPPXQHFRPSOH[HVFDQDOVREH useful in cases of paralytic rabies (3)
Introduction Laboratory techniques in rabies Fifth edition Part 1. General considerations
As rabies is an acute progressive encephalitis, the central nervous system (CNS) LVWKHPDLQWLVVXHRILQWHUHVW:KHUHDVLQWKHSDVWWKHHQWLUHEUDLQZDVUHPRYHG typically for diagnosis in animals, this cumbersome and sometimes dangerous exercise is now largely unnecessary when a full section of the brainstem is avai lable (Chapter 7 6DIHDQGVHFXUHPHWKRGVDUHDOVRVXJJHVWHGIRUREWDLQLQJDQG VKLSSLQJVDPSOHVIURPWKHĺHOGIRUXVHLQEDVLFGLDJQRVLVDQGFKDUDFWHUL]DWLRQRI pathogens (Chapter 15 %H\RQGURXWLQHQHHGVIRUHQKDQFHGODERUDWRU\EDVHG surveillance, laboratorians should also appreciate current regulatory norms for evaluation of diagnostic products available nationally or imported, human or animal vaccines, reagents used in applied research, and expectations of other future human and animal biologicals (Chapters 35 and 36 $ZLGHYDULHW\RIPHWKRGVDUHDYDLODEOHIRUXVHLQUDELHVGLDJQRVLV/\VVDYLUXV virions may be visualized directly via electron microscopy (Chapter 10 ,QIHF WLRXV}YLUXVHVDUHXVHGLQDGLYHUVLW\RISURWRFROVIURPSDWKRELRORJLFDOVWXGLHVWR WHVWVRIYDFFLQHHIĺFDF\DQGFDQEHLVRODWHGLQDQLPDOV Chapter 8) or, preferably, in tissue culture (Chapter 9 %H\RQGWUDGLWLRQDOGHPRQVWUDWLRQRIYLUDODQWLJHQV LQ WKH &16 E\ WKH ')$7 Chapter 11), an alternative is the direct rapid immu QRKLVWRFKHPLVWU\WHVW '5,7 XVLQJOLJKWPLFURVFRS\ Chapter 12 DVDVLPSOLĺ cation of its formal predecessor, traditional immunohistochemistry (Chapter 13 Antigenic typing of viral variants (Chapter 14 PD\EHSHUIRUPHGXVLQJĻXRUHVFHQW RUOLJKWPLFURVFRS\WRGLVFULPLQDWHODERUDWRU\VWUDLQVIURPĺHOGLVRODWHVDQGWR GLIIHUHQWLDWHLQIHFWHGDQLPDOVDQGKXPDQVE\YDULDQWW\SH7KH')$7PD\DOVREH SHUIRUPHGXSRQIUR]HQVNLQELRSVLHVIRUGLDJQRVLVRIKXPDQUDELHVZLWKUHVXOWV available in a few hours (Chapter 15 6HYHUDORWKHUWHVWV Chapters 16–18) have also been suggested for the detection of viral antigens, including latex agglutina tion protocols (4) ,GHDOO\DVLPSOHSRLQWRIFDUHWHVWZRXOGJUHDWO\DVVLVWLQVXUYHLOODQFHDQGULVN DVVHVVPHQWV8QIRUWXQDWHO\FXUUHQWO\DYDLODEOHOLQHDUĻRZWHVWVYDU\IURPORWWR ORWDQGDUHQRWUHFRPPHQGHGIRUPDNLQJGHFLVLRQVRQSRVWH[SRVXUHSURSK\OD[LV 3(3 LQKXPDQV The detection of viral antibodies (Chapters 19–22) is useful for several purposes, LQFOXGLQJEDVLFGLDJQRVLVULVNDVVHVVPHQWVLPPXQRJHQLFLW\VWXGLHVDQGHSLGH PLRORJLFDO LQYHVWLJDWLRQV $QWLERG\ GHWHFWLRQ PD\ EH GLVFHUQHG IXQFWLRQDOO\ E\ QHXWUDOL]DWLRQRUWKURXJKELQGLQJVWXGLHVVXFKDVHQ]\PHOLQNHGLPPXQRVRUEHQW assays (ELISAs), as detailed in earlier editions of this manual and in a recent publi cation (5)DUHQRWUHSHDWHGKHUHLQDVSURWRFROVKDYHQRWFKDQJHGFRQVLGHUDEO\ $GGLWLRQDOO\RYHU WKH SDVW IHZ \HDUV D QXPEHU RI FRPPHUFLDO (/,6$ NLWV KDYH EHFRPH DYDLODEOH 5HFHQW LQWHUQDWLRQDO WHVWLQJ KDV VKRZQ JRRG UHSURGXFLEL lity among laboratories as particularly useful for immunological insights for oral vaccination programmes against rabies in wildlife (6)*LYHQWKHRSSRUWXQLW\IRU cell culture or ELISA formats, antibody detection in animals is no longer recom mended as state of the art and is included only as a courtesy for those countries LQZKLFKLWKDVEHHQUHWDLQHGKLVWRULFDOO\XQWLOQHZHURSWLRQVPD\EHDGRSWHG %H\RQGGHPRQVWUDWLRQRIYLUDODQWLJHQVDQGDQWLERGLHVDZLGHYDULHW\RIPROH FXODU WHFKQLTXHV DUH DYDLODEOH WRGD\ IRU QXFOHLF DFLG DPSOLĺFDWLRQ DQG JHQHWLF comparisons (Chapters 27–34 :KLOHLQWKHSDVWPDQ\RIWKHVHPROHFXODUSURWR FROVZHUHXVHGLQDFRQĺUPDWRU\EDVLVWRGD\PDQ\DUHXVHGDVDVWDQGDORQH IRU SULPDU\ GLDJQRVLV %RWK FRQYHQWLRQDO DQG UHDOWLPH UHYHUVH WUDQVFULSWDVH
Introduction Laboratory techniques in rabies Fifth edition Part 1. General considerations
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References
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General considerations Chapter 2 Lyssaviruses Introduction 5DELHV LV DQ DFXWH YLUDO HQFHSKDOLWLV ZLWK D FDVH IDWDOLW\ UDWH DSSUR[LPDWLQJ ZKLFK LV WKH KLJKHVW DPRQJ NQRZQ LQIHFWLRXV GLVHDVHV 7KLV ]RRQRVLV NQRZQWRKXPDQNLQGIRUPLOOHQQLDLVDVVRFLDWHGSULPDULO\ZLWKWKHELWHVRIqPDGr dogs (1) 7KH (QJOLVK QDPH OLNHO\ RULJLQDWHV IURP WKH 6DQVNULW ZRUG qUDEKDVr PHDQLQJqYLROHQFHr$VLPLODUHW\PRORJ\H[LVWVLQRWKHUODQJXDJHV WKHQDPHRI WKHGLVHDVHW\SLĺHVXQFRQWUROOHGYLROHQFHDJJUHVVLRQPDGQHVVRUIUHQ]\ %HVLGHV qFODVVLFDOr UDELHV YLUXV 5$%9 D QXPEHU RI RWKHU SK\ORJHQHWLFDO O\UHODWHGYLUXVHVVHJUHJDWHLQWRWKHJHQXVLyssavirus as described during recent decades (2)6RPHRIWKHVHYLUXVHVDUHDQWLJHQLFDOO\DQGVHURORJLFDOO\UHODWHGWR 5$%9ZKHUHDVRWKHUVDUHPRUHGLYHUJHQWDQGDVVXFKDUHQRWFRYHUHGIXOO\E\ commercial biologicals for rabies prophylaxis (3–5)6WLOOPRUHWKDQ RIKXPDQ UDELHVFDVHVDUHFDXVHGE\LQIHFWLRQZLWK5$%9PHGLDWHGE\GRJV3RVWH[SR sure prophylaxis (PEP) virtually assures complete prevention of the disease if DGPLQLVWHUHGSURPSWO\DQGSURSHUO\DQGKDVEHHQDYDLODEOHIRURYHUDFHQWXU\ Nevertheless, rabies still causes tens of thousands of human deaths every year due to poor availability or affordability of appropriate biologicals, particularly in those areas where the demand for them is highest, such as developing countries of tropical Asia and Africa (6) As with any infectious disease, effective programmes of rabies management UHO\RQULJRURXVODERUDWRU\EDVHGVXUYHLOODQFHUREXVWGLDJQRVWLFWRROVDQGKLJKO\ HIĺFDFLRXVELRORJLFDOV7KLVODERUDWRU\PDQXDOFRYHUVWKHPRVWHVVHQWLDOPRGHUQ PHWKRGVRIUDELHVGLDJQRVLVSUHYHQWLRQDQGFRQWURO7KLVFKDSWHUUHYLHZVVDOLHQW IHDWXUHVRIO\VVDYLUXVVWUXFWXUHIXQFWLRQSDWKRELRORJ\DQGSK\ORJHQ\ Virion structure and life cycle /\VVDYLUXV YLULRQV DUH EXOOHWVKDSHG t QP ORQJ DQG t QP LQ GLDPHWHU7KH\DUHFRPSRVHGRIWZRIXQFWLRQDOXQLWV WKHLQWHUQDOQXFOHRFDSVLG 1& FRUHWKDWLQFOXGHVWKHJHQRPLF51$WLJKWO\ERXQGWRWKHQXFOHRSURWHLQ 1 SKRVSKRSURWHLQ 3 DQGYLUDOSRO\PHUDVH /IURPqODUJHSURWHLQr DQGWKHH[WHUQDO XQLWUHSUHVHQWHGE\DELOD\HUOLSLGHQYHORSH DFTXLUHGIURPKRVWFHOOPHPEUDQHV GXULQJEXGGLQJ ZLWKSURWUXGLQJVSLNHVRIWKHYLUDOJO\FRSURWHLQ * 7KHPDWUL[ SURWHLQ 0 SUHVHQWEHWZHHQWKHVHWZRXQLWVFRQGHQVHVWKH1&DQGLQWHUDFWVZLWK the G protein endodomain (7) 7KHJHQRPHLVUHSUHVHQWHGE\DQRQVHJPHQWHGOLQHDUQHJDWLYHVHQVHVLQJOH VWUDQGHG51$ VV51$ DERXWNEORQJ,WLQFOXGHVĺYHPDMRUJHQHVWKDWDUH DUUDQJHGLQWKHFRQVHUYHGOLQHDURUGHUp130*/p(DFKRIWKHLQGLYLGXDO Lyssaviruses Laboratory techniques in rabies Fifth edition Part 1. 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General considerations (15)7KHUHSOLFDWLRQSURFHVV\LHOGV1&VFRQWDLQLQJIXOOOHQJWKDQWLJHQRPHVHQVH 51$ZKLFKLQWXUQVHUYHDVWHPSODWHVIRUWKHV\QWKHVLVRIJHQRPHVHQVH51$ During their synthesis, both the nascent antigenome and the genome are encap VLGDWHGE\WKH1SURWHLQ &\WRSODVPLFLQFOXVLRQVNQRZQKLVWRULFDOO\DV1HJULERGLHV(16), can be frequently REVHUYHG E\ OLJKW PLFURVFRS\ LQ 6HOOHUVWDLQHG EUDLQ LPSUHVVLRQV 7KHVH LQFOX sions and viral antigens are detected by the direct rapid immunohistochemistry WHVW '5,7 DQG WKH GLUHFW ĻXRUHVFHQW DQWLERG\ WHVW ')$7 DQG DUH W\SLFDO IRU O\VVDYLUXVLQIHFWLRQ6XFKLQFOXVLRQVDUHIXQFWLRQDOVWUXFWXUHVLQZKLFKYLUDOWUDQV FULSWLRQDQGUHSOLFDWLRQWDNHSODFHDQGZKHUHQHRDVVHPEOHG1&VDFFXPXODWH (17)6LPXOWDQHRXVO\WKHYLUDO0DQG*SURWHLQVORFDOL]HWRWKHSODVPDPHPEUDQH 7KH0SURWHLQDFFXPXODWHVRQWKHF\WRSODVPLFVLGHRI*HQULFKHGPLFURGRPDLQV on the plasma membrane as NCs condense into tightly coiled structures by inte UDFWLQJZLWKWKH0SURWHLQ7KHPLFURGRPDLQVFRQWDLQLQJKLJKOHYHOVRI*SURWHLQ DORQJZLWKWKHFRQWLQXHGFRQGHQVDWLRQRI0513VWUXFWXUHVIDFLOLWDWHRXWZDUG membrane curvature and eventual virion budding (18)9LUDOSURJHQ\DUHUHOHDVHG DWWKHV\QDSWLFMXQFWLRQVDQGSURSDJDWHWRWKHFHQWUDOQHUYRXVV\VWHP &16 E\ XVLQJPRWRURUVHQVRU\QHXURQVIROORZLQJQHXURQDOFRQQHFWLRQV Viral pathogenesis Generally, virions are delivered into a wound via a bite or, rarely, contamination RIPXFRXVVXUIDFHVVXFKDVKLJKO\FRQFHQWUDWHGDHURVROVJHQHUDWHGLQODERUDWRU\ settings or in the unique environment of certain caves inhabited by colonies of PXOWLPLOOLRQLQGLYLGXDOEDWVLQFOXGLQJTadarida brasiliensis (19)8VLQJHLWKHUVHQVRU\ RUPRWRUQHXURQVYLUXVHVPRYHFHQWULSHWDOO\WRWKH&161HXURWURSLVPDYRLGVRU limits surveillance by the host immune system, resulting in the absence of an early DQWLERG\ UHVSRQVH 7KH GXUDWLRQ RI D QRQFOLQLFDO LQFXEDWLRQ SHULRG DYHUDJHV t}PRQWKVEXWPD\YDU\IURPDZHHNWRVHYHUDOPRQWKVRUHYHQ\HDUV(20, 21) During the incubation period, rabies diagnosis cannot be performed on animal or human tissues due to the very limited viral load, uncertain location in peripheral QHUYHVDQGWKHDEVHQFHRIDGHWHFWDEOHLPPXQHUHVSRQVH Upon delivery to the CNS, lyssaviruses disseminate rapidly and may affect QHDUO\DOOUHJLRQVRIWKH&167KHPHGXOODREORQJDWDLVWKHPDLQDUHDIURPZKLFK virus is transported to the salivary glands along innervation pathways, allowing further excretion with saliva and transmission to other susceptible hosts via a bite (22)7KHUHIRUHWKHPRVWUHOLDEOHGLDJQRVLVRIUDELHVFDQEHGRQHGXULQJWKH clinical period of the disease (or after death) on CNS tissues; the brainstem is the PRVWLPSRUWDQWUHJLRQIRUGLDJQRVWLFWHVWV9LUXVFDQEHVSUHDGFHQWULIXJDOO\IURP the CNS to peripheral tissues also via innervation pathways, allowing antemortem GHWHFWLRQRIDQWLJHQVDQGQXFOHLFDFLGVLQKDLUIROOLFOHVE\DVNLQELRSV\DWWKH QDSH RI WKH QHFN 6XFK GHSRVLWLRQ RI SHULSKHUDO WLVVXH PD\ SRVH D KD]DUG IRU scavenging mammals when they consume carcasses of rabid animals (23) and in human recipients of tissue or organ transplantation (24, 25) 7KH FOLQLFDO SHULRG RI UDELHV LV XVXDOO\ OLPLWHG WR t ZHHNV 7KH SURGURPDO SHULRGLVODFNLQJLQVSHFLĺFFOLQLFDOVLJQVZKHQSDWLHQWVGHYHORSIHYHUPDODLVH DQGVRPHWLPHVĻXOLNHV\PSWRPV$IIHFWHGVXEMHFWVPD\IHHOSDUDHVWKHVLDDQG WLQJOLQJ DW WKH LQRFXODWLRQ VLWH :LWK SURJUHVVLRQ WR HQFHSKDOLWLV FHUHEUDO VLJQV Lyssaviruses Laboratory techniques in rabies Fifth edition Part 1. General considerations PDQLIHVW+XPDQVRIWHQGHYHORSKDOOXFLQDWLRQVDQGGHOLULXP6RPHV\PSWRPV ZKLFKKDYHEHHQFRQVLGHUHGDVqFODVVLFDOrEXWDUHREVHUYHGLQRQO\œRI SDWLHQWVDUHK\GURSKRELDDHURSKRELDSKRWRSKRELDDQGSKRQRSKRELD,PPXQH UHVSRQVH FDQ XVXDOO\ EH GHWHFWHG DIWHU VHYHUDO GD\V RI GLVHDVH RQVHW $V WKH 1}SURWHLQLVSUHVHQWLQLQIHFWHGFHOOVLQODUJHDPRXQWVDQWL1DQWLERGLHV QRQQHX WUDOL]LQJ FDQEHGHWHFWHGE\VXFKPHWKRGVDVWKHLQGLUHFWĻXRUHVFHQWDQWLERG\ ,)$ WHVWRUWKHHQ]\PHOLQNHGLPPXQRVRUEHQWDVVD\ (/,6$ 7KHYLUXVQHXWUD OL]LQJDQWL*DQWLERGLHVFDQEHXVXDOO\GHWHFWHGDIHZGD\VODWHULQERWKVHUXP DQGFHUHEURVSLQDOĻXLG &6) $QWLO\VVDYLUXVDQWLERGLHVPD\EHSUHVHQWLQWKH VHUXP RI YDFFLQDWHG SDWLHQWV ZKHUHDV WKHLU SUHVHQFH LQ WKH &6) LV LQGLFDWLYH RI&16LQIHFWLRQ UDELHV ,QPRVWFDVHVKRZHYHUWKHLPPXQHUHVSRQVHFDQQRW FOHDUO\VVDYLUXVLQIHFWLRQ2QFHFOLQLFDOV\PSWRPVDSSHDUGHDWKRFFXUVXVXDOO\ ZLWKLQtGD\VGXHWRFDUGLDFRUUHVSLUDWRU\IDLOXUHDOWKRXJKLQWHQVLYHFDUHPD\ prolong the disease beyond a month (26)$IHZFDVHVRIUHFRYHU\IURPFOLQLFDO rabies have been documented in humans and animals (6, 27) Phylogeny and taxonomy 8QWLOWKH VLWZDVEHOLHYHGWKDW5$%9ZDVXQLTXHDVWKHVROHFDXVHRI UDELHV+RZHYHUIXUWKHUGLVFRYHULHVRI/DJRVEDWYLUXV /%9 DQG0RNRODYLUXV 02.9 LQ $IULFD GHPRQVWUDWHG WKH H[LVWHQFH RI RWKHU UKDEGRYLUXVHV DQWLJHQL FDOO\UHODWHGWR5$%9DQGWKHWHUPqUDELHVVHURJURXSrZDVLQWURGXFHG(28)6LPXO WDQHRXVO\DQRWKHUUDELHVUHODWHGDJHQW'XYHQKDJHYLUXV '899 ZDVGHVFULEHG from South Africa (29) %DVHG RQ VHURORJLFDO DQG DQWLJHQLF FURVVUHDFWLYLW\WKH UDELHVUHODWHG YLUXVHV ZHUH FODVVLĺHG LQWR VHURW\SHV /DWHU ZLWK WKH DGYHQW RI monoclonal antibodies for diagnostic and typing purposes, it was demonstrated WKDWqUDELHVYLUXVHVrFLUFXODWLQJLQ(XURSHDQEDWVZHUHUHODWHGWR'899DQGWKH\ were included in the same serotype (29, 30))XUWKHUULJRURXVVWXGLHVZLWKH[WHQGHG panels of monoclonal antibodies helped to distinguish between these viruses as ZHOO DV EHWZHHQ (XURSHDQ EDW YLUXVHV WKHPVHOYHV 6XFK WHUPV DV qELRW\SHVr appeared in the taxonomic literature for a limited period of time (31)+RZHYHU WKH PRVW FRPSUHKHQVLYH GLIIHUHQWLDWLRQ RI UDELHVUHODWHG YLUXVHV ZDV DFKLHYHG via sequencing and comparison of their genes (32)7KHVHODWWHUHIIRUWVLQJHQHUDO FRQĺUPHGDQGFODULĺHGĺQGLQJVIURPPRQRFORQDODQWLERG\VWXGLHVEXWFODVVLĺ cation switched from viral serotypes to genotypes, along with the establishment of the genus LyssavirusZKHUHDOOUDELHVUHODWHGYLUXVHVKDYHEHHQLQFOXGHG7KH H[LVWLQJqJHQRW\SHVrVHUYHGDVDEDVLVIRUWD[RQRP\RIO\VVDYLUXVHVIRUDOPRVW WZRGHFDGHVEXWZHUHUHĺQHGWRVDWLVI\WKHRIĺFLDOUXOHVHVWDEOLVKHGE\WKH,QWHU QDWLRQDO &RPPLWWHH RQ 7D[RQRP\RI 9LUXVHV ,&79 ZKLFK RSHUDWHV ZLWK PRUH FRPSOH[GHĺQLWLRQVVXFKDVYLUDOVSHFLHV Increased surveillance has led to the discovery of several novel lyssaviruses GXULQJ UHFHQW GHFDGHV $W SUHVHQW WKH JHQXV LQFOXGHV DW OHDVW HVWDEOLVKHG VSHFLHV DQG RQH YLUXV DZDLWLQJ ,&79 DVVHVVPHQW 7DEOH 'XULQJ WKH ,&79 LQWURGXFHG D YDULDQW RI ELQRPLDO YLUXV QRPHQFODWXUH (33) 7KLV ZLOO OLNHO\ HYROYHLQWKHIXWXUHDVLQWKHSUHVHQWIRUPLWPD\FDXVHFRQIXVLRQ HJUDELHV YLUXV>DYLUXV@EXWRabies lyssavirus>DYLUDOVSHFLHV@ 2IQRWHRQO\YLUXVQDPHVEXW QRWVSHFLHVQDPHVFDQEHDEEUHYLDWHG )LJ 7KLVPDQXDOXVHVYLUXVQDPHV when in reference to the viruses, whereas species names are used solely in the Lyssaviruses Laboratory techniques in rabies Fifth edition Part 1. General considerations VSHFLĺFWD[RQRPLFFRQWH[W,QFRQWUDVWWRRWKHUDUHDVRIELRORJ\YLUXVQDPHVDQG YLUDOVSHFLHVQDPHVFDQQRWEHXVHGDVV\QRQ\PV ,QWHUVSHFLHVJHQHWLFGLVWDQFHVEHWZHHQO\VVDYLUXVHVDUHVKRUWHUWKDQLQWHUVSH FLHV JHQHWLF GLVWDQFHV LQ RWKHU JHQHUD RI 5KDEGRYLULGDH 7R DFFRPSOLVK PRUH FRPSUHKHQVLYHGHPDUFDWLRQRIO\VVDYLUXVVSHFLHVIRXUPDMRUFULWHULDDUHXVHG L *HQHWLF GLVWDQFHV ZLWK WKH WKUHVKROG RI t QXFOHRWLGH LGHQWLW\ IRU WKHFRPSOHWH1JHQHRUtQXFOHRWLGHLGHQWLW\IRUWKHFRQFDWHQDWHG FRGLQJ UHJLRQV RI 130*/ JHQHV *OREDOO\ DOO LVRODWHV EHORQJLQJ WR the same species have higher identity values than the threshold, except WKHYLUXVHVFXUUHQWO\LQFOXGHGLQWRWKH/DJRVEDWO\VVDYLUXVVSHFLHV%DVHG RQWKHVHREVHUYDWLRQVDQGRQDSRRUFURVVQHXWUDOL]DWLRQEHWZHHQLVRODWHV from different Lagos bat virus lineages, several authors have suggested subdividing Lagos bat lyssavirus into several separate species (34, 35) (ii) Topology and consistency of phylogenetic trees, obtained with various HYROXWLRQDU\PRGHOV LLL $QWLJHQLF SDWWHUQV LQ UHDFWLRQV ZLWK DQWL1& PRQRFORQDO DQWLERGLHV DQG VHURORJLFDOFURVVUHDFWLYLW\XVLQJSRO\FORQDODQWLVHUD (iv) Additional characteristics such as ecological properties, host and geogra phical range; pathological features are considered, whenever available (7) 0RUHJOREDOO\EDVHGRQJHQHWLFGLVWDQFHVDQGVHURORJLFDOFURVVUHDFWLYLW\WKH Lyssavirus genus has been subdivided into two main phylogroups (36)7KHGHPDU FDWLRQ ZDV EDVHG RQ SK\ORJHQHWLF UHODWLRQVKLSV ł DPLQR DFLG VHTXHQFH identity within the G ectodomain between viruses of one phylogroup and }}VHTXHQFHLGHQWLW\EHWZHHQYLUXVHVIURPGLIIHUHQWSK\ORJURXSV FURVVQHX WUDOL]DWLRQ ZLWKLQ SK\ORJURXSV EXW DEVHQFH RI FURVVQHXWUDOL]DWLRQ EHWZHHQ PHPEHUVRIGLIIHUHQWSK\ORJURXSV WKHSUHVHQFHRI.5LQWKH*RISK\ORJURXS ,YLUXVHVDQGRI'LQWKH*RISK\ORJURXS,,YLUXVHVZLWKWKHIROORZLQJLPSOLFD WLRQIRUDUHGXFHGSDWKRJHQLFLW\IRUWKHODWWHU*HQHUDOO\VHURORJLFDOFURVVUHDFWL vity correlated somewhat with genetic distances, and sequences that shared more WKDQDPLQRDFLGVHTXHQFHLGHQWLW\ZLWKLQWKHLU*HFWRGRPDLQVFURVVQHXWUD lized each other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ĺFLHQW protection against phylogroup I lyssaviruses but not against other members of the JHQXVSDUWLFXODUO\WKRVHO\VVDYLUXVHVLQSK\ORJURXS:&%9,.29DQG//(%9}(3, 36–38)0RUHRYHUDQWLJHQLFDQGJHQHWLFGLVWLQFWLRQVVKRXOGEHFRQVLGHUHGLQDSSOL cation of any diagnostic tests to ensure their ability to detect all divergent lyssa YLUXVHVDVGHVFULEHGLQWKHUHOHYDQWFKDSWHUVRIWKLVPDQXDO Lyssaviruses Laboratory techniques in rabies Fifth edition Part 1. General considerations 7DEOH9LUXVHVFXUUHQWO\LQFOXGHGLQWKHJHQXVLyssavirus Recognized Geographical and proposed Phylogroup Natural host Comments range species Rabies lyssavirus I %DWV &KLURSWHUD Terrestrial mammals 5HVSRQVLEOHIRUPRVWFDVHV (type species) of multiple species, – worldwide (except RIKXPDQUDELHVLQWKHZRUOG terrestrial mammals Australia, Antarctica All currently available human (predominantly and several insular and veterinary vaccine strains Carnivora) territories); bats – originate from viruses of this New World only VSHFLHV Australian bat I Pteropodid bats (at Australia (perhaps, Given limited surveillance, host lyssavirus least four species with several range among insectivorous bats of Pteropus genus) surrounding islands) PD\EHJUHDWHU7KUHHKXPDQ and insectivorous FDVHVGRFXPHQWHG bats Saccolaimus albiventris) European bat I Insectivorous bats 7KHPDMRUSDUWRI Given the limited surveillance in lyssavirus,W\SH (predominantly Europe, from Spain to eastern Europe and Asia, may Eptesicus serotinus) WKH8NUDLQH be distributed more broadly, along the reservoir species UDQJH6SLOORYHULQIHFWLRQVLQ wild and companion animals, as well as a human case have been GRFXPHQWHG European bat I Insectivorous bats 1RUWKZHVWHUQ Two human cases have been lyssavirusW\SH (predominantly Myotis Europe GRFXPHQWHG daubentonii and Myotis dasycneme) Khujand lyssavirus I Insectivorous bat Central Asia .QRZQE\DVLQJOHLVRODWH Myotis mystacinus Given the limited surveillance in eastern Europe and Asia, may EHGLVWULEXWHGPRUHEURDGO\ No human cases have been GRFXPHQWHG Aravan lyssavirus I Insectivorous bat Central Asia .QRZQE\DVLQJOHLVRODWH Myotis blythii Given the limited surveillance in eastern Europe and Asia, may EHGLVWULEXWHGPRUHEURDGO\ No human cases have been GRFXPHQWHG Bokeloh bat I Insectivorous bat Europe Known by three isolates from lyssavirus Myotis nattereri :HVWHUQ(XURSH1RKXPDQ FDVHVKDYHEHHQGRFXPHQWHG Irkut lyssavirus I Insectivorous bats Eastern Asia Known by three isolates, from from Murina genus bats and from a human who GHYHORSHGUDELHVDIWHUDEDWELWH Duvenhage I Insectivorous bats 6XE6DKDUDQ$IULFD Known by four isolates, three of lyssavirus which came from humans, bitten by bats, and one from a bat, of the presumably Miniopterus VSHFLHV Lyssaviruses Laboratory techniques in rabies Fifth edition Part 1. General considerations 7DEOH&RQWLQXHG Recognized Geographical and proposed Phylogroup Natural host Comments range species Lagos bat II Pteropodid 6XE6DKDUDQ$IULFD Includes several lineages lyssavirus bats of several ZLWKORQJJHQHWLFGLVWDQFHV genera (Eidolon Potentially, in the future may be helvum, Rousettus VXEGLYLGHGLQWRtVHSDUDWH aegyptiacus, VSHFLHV6SLOORYHULQIHFWLRQV EpomophorusVSS reported in wild and companion HWF DQLPDOV1RKXPDQFDVHV GRFXPHQWHG Mokola lyssavirus II 8QNQRZQ 6XE6DKDUDQ$IULFD Twice isolated from shrews, once IURPDURGHQW7KHPDMRULW\RI other isolates were obtained from companion animals, such as cats and dogs, as the result of VSLOORYHULQIHFWLRQV7ZRKXPDQ FDVHVKDYHEHHQUHSRUWHG Shimoni bat II Insectivorous bat Kenya .QRZQE\DVLQJOHLVRODWH lyssavirus Hipposideros Serological surveys suggest that commersoni (H. Hipposideros commersoni (H. vittatus) vittatus LVWKHOLNHO\UHVHUYRLU No human cases have been GRFXPHQWHG West Caucasian Insectivorous 6RXWKHDVWHUQ Known by a single isolate from bat lyssavirus bats from genus Europe, probably WKH&DXFDVLDQUHJLRQ+RZHYHU Miniopterus Africa serological surveys suggest WKDWWKLVRURWKHUVHURORJLFDOO\ related virus may be present in Miniopterus bats in Africa .HQ\D 1RKXPDQFDVHVKDYH EHHQGRFXPHQWHG Ikoma lyssavirus African civet 8QLWHG5HSXEOLFRI .QRZQE\DVLQJOHLVRODWH7KH Civettictis civetta Tanzania QDWXUDOKRVWLVTXHVWLRQDEOH Given phylogenetic relatedness to the West Caucasian bat virus, the index case in African civet PD\UHVXOWIURPDVSLOORYHU LQIHFWLRQRIEDWRULJLQ1RKXPDQ FDVHVKDYHEHHQGRFXPHQWHG Lleida bat Insectivorous 6SDLQDQG)UDQFH .QRZQE\DVLQJOHLVRODWH lyssavirus bat Miniopterus >XQSXEOLVKHG No human cases have been schreibersii GDWD@ GRFXPHQWHG Gannoruwa bat I ,QGLDQĻ\LQJIR[ 6UL/DQND Several isolates obtained from lyssavirus Pteropus medius the single bat species in Sri (P. giganteus) /DQND*LYHQWKHKRVWUDQJH may be present in the Indian subcontinent and surrounding DUHDV1RKXPDQFDVHVKDYH EHHQGRFXPHQWHG Taiwan bat I Insectivorous bat Taiwan, China .QRZQE\WZRLVRODWHV lyssavirus Pipistrellus abramus Given the host range, may be distributed broadly in eastern $VLD1RKXPDQFDVHVKDYH EHHQGRFXPHQWHG Lyssaviruses Laboratory techniques in rabies Fifth edition Part 1. General considerations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yssaviruses Laboratory techniques in rabies Fifth edition Part 1. General considerations Distribution, evolution and host range Lyssaviruses are distributed globally except in Antarctica and several insular LVODQGV,QWKHODWWHULQWURGXFWLRQRIWKHLQIHFWLRQLVVWLOOSRVVLEOHZLWKPLJUDWRU\ bats, or via human activity as happened with the introduction of rabid dogs followed by host switching to populations of mongooses (introduced by humans as well) in Grenada (39)DQG3XHUWR5LFR (40), or the introduction of rabid ferret badgers to Taiwan (41) $OWKRXJK DOO ZDUPEORRGHG DQLPDOV LQFOXGLQJ ELUGV (42), are susceptible, PDPPDOVIURPWKHRUGHUV&KLURSWHUD EDWV DQG&DUQLYRUDDUHWKHPDMRUQDWXUDO reservoirs in which lyssaviruses perpetuate and evolve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hosts of the virus (44) In addition, all lyssavirus diversity has been documented in the Old World but RQO\5$%9ZDVGHWHFWHGLQWKH1HZ:RUOG,WLVUHPDUNDEOHWKDWLQWKH1HZ:RUOG EDWVRIGLIIHUHQWVSHFLHVPDLQWDLQFLUFXODWLRQRIPXOWLSOH5$%9OLQHDJHVZKHUHDV LQWKH2OG:RUOGEDWVKDUERXUDYDULHW\RIGLYHUJHQWO\VVDYLUXVHVEXWQRW5$%9 Such discrepancies compromise the commonly accepted theory that lyssaviruses HYROYHGLQEDWVDQGWKDWDWOHDVWWKHLQLWLDOHYROXWLRQWRRNSODFHLQWKH2OG:RUOG OLNHO\LQ$IULFDZKHUHWKHPDMRULW\RIGLYHUJHQWO\VVDYLUXVHVDUHSUHVHQW(43),Q}SDUWL FXODULIZHDFFHSWWKHHVWLPDWLRQVRIO\VVDYLUXVDJHEDVHGRQPROHFXODUFORFN WHFKQLTXHVZKLFKLQGLFDWHWKDWPRVW5$%9OLQHDJHVHPHUJHGWHQVWRKXQGUHGVRI \HDUVDJRWKHQWKHHQWLUHJHQXVLVQRROGHUWKDQt\HDUV(45–47) An increasing amount of evidence suggests that timescale estimation performed on a limited set of recently sampled sequences cannot provide realistic inferences for viruses evolving under constraints of purifying selec WLRQ}(48)7KLVLVH[DFWO\WKHFDVHIRUO\VVDYLUXVHVWKDWHYROYHQHXWUDOO\XQGHUVWURQJ purifying selection constraints (49),QJHQHUDOHYROXWLRQDU\UDWHVREVHUYHGLQDVHW of sequences exhibit time dependency and increase towards the present because RIWKHWUDQVLHQWPXWDWLRQV\HWWREHUHPRYHGE\SXULI\LQJVHOHFWLRQ6XEVWLWXWLRQ rates estimated during long time frames will therefore be lower systematically WKDQ WKRVH REWDLQHG GXULQJ VKRUW WLPH IUDPHV 7KLV HIIHFW LV DV VWURQJ DV RU stronger than, purifying selection, which shapes virus evolution (50, 51),QRWKHU words, timescale estimations based on substitution rates are useful only for the time frame encompassed by the sampling period, but cannot be extrapolated IRUORQJHUSHULRGVRIWLPH)RUH[DPSOHDQDOWHUQDWLYHDSSURDFKZLWKLPSURYHG substitution saturation, increased the age of coronaviruses by several orders of PDJQLWXGHFRPSDUHGZLWKSUHYLRXVPROHFXODUFORFNHVWLPDWHVDQGWKHUHVXOWLQJ timescale was comparable to that of the coronavirus reservoir hosts (52)3HUKDSV lyssaviruses should be considered more ancient pathogens that may have evolved in bats during the Cretaceous period in the territory of modern Africa (the region IURPZKLFKWKHEDWVRULJLQDWHG DQGWKDWDQDQFLHQW5$%9IROORZHGEDWGLVSHUVLRQ Lyssaviruses Laboratory techniques in rabies Fifth edition Part 1. General considerations WR6RXWK$PHULFDtPLOOLRQ\HDUVDJRYLDLVODQGKRSSLQJDQGLQWHUFRQWLQHQWDO ĻLJKW}(53)7KLV}K\SRWKHVLV(54)PLJKWH[SODLQDQLQGHSHQGHQWHYROXWLRQRI5$%9 in New World bats and the absence of the virus in related taxa of Old World bats HJIURPJHQHUDEptesicus, Myotis 6XFK1HZ:RUOGEDWYLUXVHVIXUWKHUVKLIWHG WR FDUQLYRUHV LQFOXGLQJ VNXQNV DQG UDFFRRQV ZKLFK DUH SDUWV RI WKH qLQGLJH QRXV$PHULFDQ5$%9OLQHDJHrDQGDUHZHOOGLVWLQJXLVKHGIURPDOO5$%9OLQHDJHV FLUFXODWLQJLQWKH2OG:RUOGDQGIURPWKHqFRVPRSROLWDQrFDQLQH5$%9ZKLFK is dispersed globally, with rabid dogs accompanying human migrations during recent centuries (55, 56) 8VXDOO\ HDFK O\VVDYLUXV SK\ORJHQHWLF OLQHDJH LV DVVRFLDWHG ZLWK D VSHFLĺF KRVW7KHVHUHODWLRQVKLSVDUHEHVWVWXGLHGIRU5$%9ZKLFKLVGLVWULEXWHGJOREDOO\ 7KH}QXPEHURISK\ORJHQHWLFOLQHDJHVRI5$%9GRFXPHQWHGLQ1HZ:RUOGEDWV FRUUHVSRQGVURXJKO\WRWKHQXPEHURIEDWVSHFLHVWHVWHG0RUHRYHUVRPHEURDGO\ distributed bat species, such as Eptesicus fuscus (the big brown bat) and Tadarida brasiliensis WKH0H[LFDQIUHHWDLOHGEDW KDUERXUVHYHUDOOLQHDJHVRI5$%9(47, 57) $PRQJ ZLOG FDUQLYRUHV VNXQNV RI VHYHUDO GLIIHUHQW VSHFLHV DSSHDU KLJKO\ SHUPLVVLYHWRYDULRXV5$%9YDULDQWV,QDGGLWLRQWRWKHVRXWKFHQWUDODQG0H[LFDQ VNXQN5$%9VZKLFKDUHDVVRFLDWHGSK\ORJHQHWLFDOO\ZLWKEDWYLUXVHVWKHUHDUH VHYHUDORWKHUOLQHDJHV LHQRUWKFHQWUDOVNXQN&DOLIRUQLDVNXQNDQGRWKHUVNXQN OLQHDJHVIURP0H[LFR ZKLFKEHORQJWRDqFRVPRSROLWDQr5$%9OLQHDJH6NXQNV LQ HDVWHUQ &DQDGD KDYH DFTXLUHG WKH $UFWLF 5$%9 YDULDQW (55, 56) $GGLWLRQDOO\ VNXQNV DORQJ WKH HDVW FRDVW RI 1RUWK $PHULFD DUH IUHTXHQWO\ LQIHFWHG E\ WKH UDFFRRQ5$%9(58, 59) Conversely, Procyon lotor (the North American raccoon) seems well adapted DQG FRQVWUDLQHG RQO\ WR RQH VSHFLĺF 5$%9 YDULDQW ZKLFK LV EHOLHYHG WR KDYH HPHUJHGGXULQJWKH VLQ)ORULGD86$DQGWKHUHDIWHUKDVUDSLGO\VSUHDGDORQJ the eastern coast of North America (60, 61) 2WKHU$PHULFDQOLQHDJHVRI5$%9FLUFXODWLQJLQGRJVFR\RWHV Canis latrans) DQGVHYHUDOVSHFLHVRIIR[HVEHORQJWRWKHqFRVPRSROLWDQrJURXS0DQDJHPHQW RIFDQLQHUDELHVUHVXOWHGLQHOLPLQDWLRQRIGRJPHGLDWHGUDELHVLQWKH86$DQG &DQDGDDOWKRXJK5$%9VWLOOFLUFXODWHVLQVRPHSDUWVRI/DWLQ$PHULFD0RQJRRVHV LQVHYHUDO&DULEEHDQLVODQGVDFTXLUHGWKHqFRVPRSROLWDQr5$%9IURPGRJVDQG maintain its circulation (56)2UDOUDELHVYDFFLQDWLRQRIZLOGOLIHVLJQLĺFDQWO\UHGXFHG 5$%9FLUFXODWLRQLQWKHLUSRSXODWLRQVLQ1RUWK$PHULFD,QDGGLWLRQWRWKHUHSUH VHQWDWLYHVRIqFRVPRSROLWDQr5$%9OLQHDJH$UFWLF5$%9FLUFXODWHVDPRQJ$UFWLF foxes (Vulpes lagopus), red foxes (V. vulpes DQGVWULSHGVNXQNV Mephitis mephitis) in northern territories of the continent (62, 63) ,Q WKH 2OG :RUOG 5$%9 KDV EHHQ GRFXPHQWHG LQ FDUQLYRUHV RQO\ ZLWK WKH H[FHSWLRQ RI HSL]RRWLFV LQ JUHDWHU NXGX DQWHORSHV Tragelaphus strepsiceros) in southern Africa (64), predominantly in canids and herpestides (65)7KHqFRVPR SROLWDQrOLQHDJHRI5$%9FLUFXODWHVLQGRJVDQGZLOGFDQLGVLQYDVWWHUULWRULHVRI Eurasia and Africa (55, 56),QDGGLWLRQGLVWLQFWqDXWRFKWKRQRXVrOLQHDJHVRI5$%9 FLUFXODWHLQGRJVLQVHYHUDODUHDVRI$IULFDDQGVRXWKHDVW$VLD$UFWLF5$%9FLUFX ODWHVFLUFXPSRODUO\DPRQJ$UFWLFDQGUHGIR[HV3K\ORJHQHWLFLQIHUHQFHVVXJJHVW WKDWWKH$UFWLF5$%9OLQHDJHRULJLQDWHGIURPVRFDOOHG$UFWLFOLNHOLQHDJHVZKLFK are present in the Indian subcontinent and in central and eastern Asia (63, 66) 5DFFRRQGRJV Nyctereutes procyonoides), besides maintaining the circulation of $UFWLFOLNH5$%9LQWKHLUQDWLYHKDELWDWVLQHDVWHUQ$VLDUHDGLO\SDUWLFLSDWHLQFLUFX Lyssaviruses Laboratory techniques in rabies Fifth edition Part 1. General considerations ODWLRQRIqFRVPRSROLWDQr5$%9LQDUHDVHQ]RRWLFIRUIR[UDELHVZKHUHWKLVVSHFLHV ZDVVXFFHVVIXOO\LQWURGXFHG7KHZROI Canis lupus) was believed historically to be DPDMRUZLOGOLIHUDELHVKRVWLQ(XUDVLD KRZHYHUUHGXFWLRQVLQLWVSRSXODWLRQKDYH GLPLQLVKHGWKHVLJQLĺFDQFHRIWKLVVSHFLHVLQWUXHFLUFXODWLRQRI5$%9(67, 68) )ROORZLQJWKHUDWLRQDOXVHRIRUDOYDFFLQDWLRQSURJUDPPHVUDELHVZDVHOLPL QDWHG IURP WKH UHG IR[ SRSXODWLRQV LQ :HVWHUQ (XURSH E\ WKH HQG RI WKH WK century (69); nevertheless, the disease is still present in red foxes, corsac foxes, MDFNDOVDQGUDFFRRQGRJVLQ(DVWHUQ(XURSHDQG$VLDZKLFKRIIHUDULVNRIUHLQ WURGXFWLRQRIWKHGLVHDVHWRWKHFRXQWULHVWKDWDUHFRQVLGHUHGqUDELHVIUHHr(65) Lyssaviruses of other species have more limited geographical and host ranges WKDQ5$%9 7DEOH )RUH[DPSOHVHYHUDOOLQHDJHVRI/%9KDYHEHHQGRFX PHQWHGLQSWHURSRGLGEDWVLQVXE6DKDUDQ$IULFD7KHHOXVLYH'899NQRZQE\ VHYHUDOLVRODWHVIURPKXPDQVELWWHQE\LQVHFWLYRURXVEDWVDQGIURPXQLGHQWLĺHG insectivorous bats, circulates in the same geographical region (70, 71)7KH}UHVHU YRLUKRVWIRU02.9LVVWLOOXQNQRZQ6SRUDGLFLVRODWLRQVIURPGRJVDQGFDWVDUH FRQVLGHUHGVSLOORYHUHYHQWVDQGWKHLQLWLDOLVRODWLRQVIURPVKUHZVUHSRUWHGGXULQJ WKH VKDYHQRWEHHQUHSHDWHGVLQFHWKDWWLPH6HURORJLFDOO\02.9FURVV UHDFWVZLWK/%9PDNLQJGLIĺFXOWPDMRULQIHUHQFHVIURPVHURORJLFDOVXUYH\VDORQH (44):&%9KDVEHHQLVRODWHGRQO\RQFHLQWKH&DXFDVXVDUHDIURPDMiniopterus schreibersii bat, but antibodies neutralizing this virus (and not neutralizing a panel of other lyssaviruses) have been detected in MiniopterusVSSEDWVIURP.HQ\D (72) 7KH//(%9QXFOHLFDFLGKDVEHHQGHWHFWHGLQDM. schreibersii bat in Spain (73) ,.29 KDV EHHQ LVRODWHG RQFH IURP DQ $IULFDQ FLYHW LQ WKH 8QLWHG 5HSXEOLF RI 7DQ]DQLDZKLFKFRXOGEHDVSLOORYHULQIHFWLRQJLYHQLWVSK\ORJHQHWLFUHODWHGQHVV WR:&%9DQG//(%9(5) (XURSHDQEDWO\VVDYLUXVHVW\SHV (%/9 DQG (%/9 KDYHEHHQGRFX PHQWHGLQVHYHUDOFRXQWULHVRI(XURSH7KHSULQFLSDOUHVHUYRLURI(%/9LVWKH serotine bat (Eptesicus serotinus ZKHUHDVWKDWRI(%/9DSSHDUVWREHEDWVIURP the Myotis genus, particularly M. daubentonii and M. dasycneme (74)$VDOORIWKHVH EDWVSHFLHVDUHGLVWULEXWHGTXLWHEURDGO\LQ(XUDVLDFLUFXODWLRQRI(%/9DQG could correspond to their broader host ranges but has gone undetected to date due to limited surveillance (75) 'HVSLWH H[WHQVLYH DOEHLW SDVVLYH VXUYHLOODQFH DQRWKHU(XURSHDQEDWO\VVDYLUXV%%/9 %RNHORKEDWO\VVDYLUXV ZDVGLVFRYHUHG RQO\GXULQJ (76)$IHZ%%/9LVRODWHVIURP1DWWHUHUpVEDWV Myotis nattereri) KDYHEHHQGRFXPHQWHGLQ*HUPDQ\)UDQFHDQGDOVRPRUHUHFHQWO\LQ3RODQG(77) 7ZREDWO\VVDYLUXVHV$5$9 $UDYDQYLUXV DQG.+89 .KXMDQGYLUXV RQHLVRODWH of each, have been obtained in Central Asia, from bats of the MyotisJHQXV}(78) $QRWKHUEDWO\VVDYLUXV,UNXWYLUXV ,5.9 KDVEHHQLVRODWHGWZLFHIURPEDWVRIWKH Murina genus in Eastern Siberia and China (79, 80) and from a human bitten by an XQLGHQWLĺHGEDWLQWKHHDVWHUQ5XVVLDQ)HGHUDWLRQ(81)$OWKRXJKVHYHUDOUHSRUWV on serological surveillance have suggested circulation of lyssaviruses among bats in South Eastern Asia (82–84), and at least two papers have described detec WLRQRIqUDELHVYLUXVrIURPEDWVLQ,QGLD (85) and Thailand (86), no isolates were DYDLODEOHIRUFKDUDFWHUL]DWLRQXQWLOtZKHQVHYHUDOGHDGDQGPRULEXQG ,QGLDQĻ\LQJIR[HV Pteropus mediusDOVRNQRZQDVP. giganteus LQ6UL/DQNDZHUH GLDJQRVHGZLWKDQRYHOYLUXV*DQQRUXZDEDWO\VVDYLUXV *%/9 ZKLFKLVSK\ORJH QHWLFDOO\UHODWHGWR$%/9WKH$XVWUDOLDQEDWO\VVDYLUXV(87))LQDOO\UHFHQWUHSRUWV account for isolation of a lyssavirus from an insectivorous bat (Pipistrellus abramus) Lyssaviruses Laboratory techniques in rabies Fifth edition Part 1. General considerations LQ7DLZDQ&KLQDGXULQJt(88, 89),Q$XVWUDOLD$%/9KDVEHHQLVRODWHG or serologically detected in several species of pteropodid bats and viruses from a SK\ORJHQHWLFDOO\GLVWLQFW$%/9OLQHDJHLVRODWHGIURPWKHLQVHFWLYRURXVEDWSacco- laimus albiventris (90, 91)6HURORJLFDOVXUYHLOODQFHKDVVXJJHVWHGWKDWO\VVDYLUXVHV UHODWHGWR$%/9PD\FLUFXODWHLQ6RXWK(DVW$VLDPRUHH[WHQVLYHO\(92) Applications to disease management The design of surveillance systems for detection and applicable testing of lyssaviruses should be based in part upon the primary intent of each programme, the available resources and personnel and the staging of disease prevention and FRQWUROIURPDQHYLGHQFHEDVHGSHUVSHFWLYH(93))RUH[DPSOHLQFRXQWULHVDWWKH early stage of canine rabies elimination, an essential goal should focus upon rele YDQWULVNDVVHVVPHQWVDQGWKHUDSLGGLDJQRVLVRIDQLPDOVWKDWH[SRVHKXPDQV SUHIHUDEO\ E\ SRVWPRUWHP GHWHFWLRQ RI DQWLJHQV LQ EUDLQ WLVVXH $QWHPRUWHP methods would be appropriate for humans with a suspect encephalitis, espe FLDOO\DIWHUDNQRZQUHFHQWH[SRVXUHWRDVXVSHFWDQLPDO9LUDODQWLERGLHVPD\EH GHWHFWHGLQVHUDDQG&6)YLUDODQWLJHQVLQVNLQELRSV\VDPSOHVDQGYLUDODPSOL FRQVLQVDOLYDDQGUHODWHGLQIHFWLRXVĻXLGV,QFDVHVRIDFXWHKXPDQGHDWKIURP suspect viral encephalitis, electron microscopy and immunohistochemistry at a QDWLRQDOUHIHUHQFHFHQWUHPD\EHEHQHĺFLDO (94))RUORFDOLWLHVIXUWKHUDGYDQFHG towards zero human fatalities from rabies mediated by dogs and thus concerned with broader pathogen discovery and detection of novel lyssaviruses, serological screening may be useful as a preliminary tool to detect viral antibodies asso ciated with various mammalian taxa, such as bats, recognizing that successful introspection is dependent upon the correct choice of viruses or viral antigens as targets (95)7KHUHDIWHUDIRFXVRQFOLQLFDOO\LOOPDPPDOV LHEDVHGRQORFDO UHSRUWV PRULEXQG LHDWEDWURRVWV RUWKRVHIRXQGIUHVKO\GHDG LHURDGNLOOHG DQLPDOV LVPRUHSURGXFWLYHDQGHIĺFLHQWWKDQFROOHFWLRQRIQRUPDOVXEMHFWV(87) Characterization of lyssaviruses circulating in a particular country or region should utilize appropriate panels of monoclonal antibodies for antigenic typing or UHOHYDQWSURWRFROVRIPROHFXODUWHFKQLTXHVVXFKDVUHDOWLPHSRO\PHUDVHFKDLQ UHDFWLRQ 3&5 DQGIXOOJHQRPLFVHTXHQFLQJ(96))URPDJOREDOSHUVSHFWLYHDVVLV tance from WHO Collaborating Centres, twinning exercises with OIE reference laboratories and engagement with regional academic or government centres of H[FHOOHQFHVNLOOHGLQDSSURSULDWHWHFKQLFDODSSURDFKHVWRO\VVDYLUXVVXUYHLOODQFH detection and characterization, is readily available, regardless of the stage in a SDWKWRZDUGVKXPDQUDELHVHOLPLQDWLRQJRDOV Lyssaviruses Laboratory techniques in rabies Fifth edition Part 1. General considerations References 7DUDQWROD$)RXUWKRXVDQG\HDUVRIFRQFHSWVUHODWLQJWRUDELHVLQDQLPDOVDQG KXPDQVLWVSUHYHQWLRQDQGLWVFXUH7URS0HG,QIHFW'LV GRL WURSLFDOPHG $PDUDVLQJKH*.%½R<%DVOHU&)%DYDUL6%HHU0%HMHUPDQ1HWDO7D[R nomy of the order Mononegavirales XSGDWH$UFK9LURO t GRL V +DQORQ&$.X]PLQ,9%ODQWRQ-':HOGRQ:&0DQDQJDQ-65XSSUHFKW&( (IĺFDF\ RI UDELHV ELRORJLFV DJDLQVW QHZ O\VVDYLUXVHV IURP (XUDVLD 9LUXV 5HV t +RUWRQ'/0F(OKLQQH\/00DUVWRQ'$:RRG-/5XVVHOO&$/HZLV1HW DO 4XDQWLI\LQJ DQWLJHQLF UHODWLRQVKLSV DPRQJ WKH O\VVDYLUXVHV - 9LURO tGRL -9, +RUWRQ'/%DQ\DUG$&0DUVWRQ'$:LVH(6HOGHQ'1XQH]$HWDO$QWLJHQLF DQGJHQHWLFFKDUDFWHUL]DWLRQRIDGLYHUJHQW$IULFDQYLUXV,NRPDO\VVDYLUXV-*HQ 9LURO tGRL YLU :+2([SHUW&RQVXOWDWLRQRQ5DELHVWKLUGUHSRUW*HQHYD :RUOG+HDOWK2UJD QL]DWLRQ :+2 7HFKQLFDO 5HSRUW 6HULHV 1R KWWS DSSVZKRLQW LULVELWVWUHDPKDQGOH HQJSGI DFFHVVHG 0D\ 'LHW]JHQ5*&DOLVKHU&+.XUDWK*.X]PLQ,95RGULJXH]//6WRQH'0HW DO)DPLO\5KDEGRYLULGDH,Q .LQJ$04$GDPV0-&DUVWHQV(%/HINRZLW](/ HGLWRUV9LUXVWD[RQRP\ QLQWKUHSRUWRIWKH,QWHUQDWLRQDO&RPPLWWHHRQ7D[RQRP\ RI9LUXVHV2[IRUG 8. 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General considerations Aerosols Since airborne rabies virus infection has been demonstrated in nature and in the laboratory, all operations which have a potential to cause aerosols should EHSHUIRUPHGZLWKLQDFODVV,,%6&WKDWLVFHUWLĺHGDQQXDOO\+LJKVSHHGPL[LQJ SURFHGXUHVVKRXOGEHFRQGXFWHGLQWLJKWO\FORVHGFRQWDLQHUVZLWKJDVNHWVZLWKLQ DQDSSURSULDWH%6&DQGKLJKVSHHGFHQWULIXJDWLRQVKRXOGEHFRQGXFWHGXVLQJ aerosol containment carriers that are only opened and manipulated under the %6& )LJ% 2WKHURSHUDWLRQVWKDWPLJKWJHQHUDWHDHURVROVVXFKDVSLSHWWLQJ DQGWLVVXHKRPRJHQL]DWLRQVKRXOGDOVREHFRQGXFWHGZLWKLQD%6&3LSHWWLQJE\ PRXWKVKRXOGEHSURKLELWHG Disinfectants 4XDWHUQDU\ DPPRQLXP GLVLQIHFWDQWV LQ GLOXWLRQ DOFRKRO HWKDQRO RULVRSURSDQRO VRDSVROXWLRQtLRGLQHVROXWLRQVDQGR[LGL]LQJDJHQWV inactivate lyssaviruses within minutes (8, 21)$GGLWLRQDOFRQWDFWWLPHVDUHUHTXLUHG to inactivate virus on materials, instruments or surfaces heavily contaminated with proteins found in cell culture media and tissues (1, 22, 23)&RPPHUFLDOTXDWHUQDU\ ammonium disinfectants, iodophors and oxidizing agents should be diluted accor ding to the registered label for hard surface decontamination (1, 21))RUSLSHWWH UHFHSWDFOHVD GLOXWLRQRIDTXDWHUQDU\DPPRQLXPFRPSRXQGDQ\LRGLQH GLVLQIHFWDQWZLWKUHVLGXDODYDLODEOHLRGLQHRIDWOHDVW RUDFRQFHQWUD WLRQRIVRDS\ZDWHURUGHWHUJHQWFDQEHXVHGEHIRUHDXWRFODYLQJ7KHVROXWLRQ VKRXOGEHDXWRFODYHGDQGGLVFDUGHGDIWHUHDFKXVH+RWVRDS\ZDWHUGHWHUJHQWV RU GLOXWHG EOHDFK VROXWLRQV FDQ EH XVHG WR FOHDQ ĻRRUV DQG ODERUDWRU\ WDEOHV Standard operating procedures for containing and cleanup of spills should be LQFOXGHGLQWKHVLWHVSHFLĺFO\VVDYLUXVELRVDIHW\PDQXDODQGWUDLQLQJSURYLGHGRQ WKHVHSURFHGXUHVWRDOOSHUVRQQHO,QFLGHQWUHFRUGVIRUVSLOOVDQGRWKHUKD]DUGRXV RFFXUUHQFHVVKRXOGEHPDLQWDLQHG Glassware, plasticware and instruments 3URSHUVXSSOLHVVKRXOGEHVHOHFWHGWRPLQLPL]HWKHRSSRUWXQLW\IRULQMXU\E\ VKDUSV :KHQ SRVVLEOH DOWHUQDWLYHV WR JODVVZDUH VXFK DV SODVWLFV VKRXOG EH VXEVWLWXWHG 6KDUSV VKRXOG EH GLVFDUGHG LQWR SURSHU FRQWDLQHUV RU DSSURSULDWH receptacles containing one of the disinfectants mentioned above, and autoclaved EHIRUHUHXVHRUGLVSRVDO Carcasses and animal tissues Carcasses and animal tissues are best disposed of within biohazard bags and WKHQLQFLQHUDWHG5DELHVYLUXVPD\EHWUDQVPLWWHGRUDOO\LQODERUDWRU\DQLPDOVZKHQ raw brain tissue containing virus is consumed (12)$OWKRXJKWKRURXJKFRRNLQJZLOO LQDFWLYDWHYLUXVFRQVXPSWLRQRIPHDWIURPDQLPDOVVXFKDVOLYHVWRFNVXEPLWWHG WRWKHGLDJQRVWLFODERUDWRU\VKRXOGEHSURKLELWHG Biosafety Laboratory techniques in rabies Fifth edition Part 1. General considerations Treatment of wounds $OOZRXQGVVKRXOGEHZDVKHGLPPHGLDWHO\DQGWKRURXJKO\IRUDWOHDVWt PLQZLWKVRDSDQGZDWHU:DVKLQJVKRXOGEHJHQWOHWRDYRLGIXUWKHUWUDXPDWL]D WLRQRIWKHWLVVXHV6XWXULQJRIZRXQGVVKRXOGEHGHOD\HGIRUDVORQJDVSRVVLEOH IURP VHYHUDO KRXUV XS WR GD\V 3XQFWXUH ZRXQGV VKRXOG EH SUREHG JHQWO\ IRU GHEULV WDNLQJ FDUH WR PLQLPL]H IXUWKHU WUDXPD /RFDO LQĺOWUDWLRQ RI ZRXQGV should be treated with rabies immunoglobulin in the naive patient (although all VWDIIZRUNLQJLQWKHUDELHVODERUDWRU\VKRXOGKDYHUHFHLYHGSUHH[SRVXUHYDFFLQD WLRQ $GGLWLRQDOPHDVXUHVVXFKDVDGPLQLVWUDWLRQRIDQWLPLFURELDOVRUDQWLWHWDQXV SURFHGXUHVZKHQLQGLFDWHGVKRXOGIROORZORFDOPHGLFDOJXLGDQFH Pre-exposure vaccination 3UHH[SRVXUHYDFFLQDWLRQVKRXOGEHUHTXLUHGIRUDOOSHUVRQVDWULVNRIH[SR VXUH VXFK DV ODERUDWRU\ VWDII ZRUNLQJ ZLWK O\VVDYLUXVHV 6XFK LPPXQL]DWLRQ should preferably consist of at least three intramuscular or intradermal doses of WLVVXHFXOWXUHUDELHVYDFFLQHRIDSRWHQF\RIDWOHDVWLQWHUQDWLRQDOXQLWV ,8 SHUGRVH9DFFLQDWLRQVKRXOGRFFXUDFFRUGLQJWRWKHSURGXFWODEHORUE\DVFKH GXOHDVUHFRPPHQGHGE\WKHFXUUHQW:+2([SHUW&RQVXOWDWLRQRQ5DELHV}(18) 7KH} SUHVHQFH RI YLUXVQHXWUDOL]LQJ DQWLERGLHV 91$ LQ YDFFLQDWHG LQGLYLGXDOV VKRXOGEHDVFHUWDLQHGZKHUHIHDVLEOHXVLQJVHUXPVDPSOHVFROOHFWHGtZHHNV DIWHUWKHODVWYDFFLQHGRVH %DVHGRQVHURORJLFDOVXUYHLOODQFHSHULRGLFERRVWHULQMHFWLRQVRIYDFFLQHDUH UHFRPPHQGHGIRUSHUVRQVDWFRQWLQXLQJULVNRIH[SRVXUHWRYLUXV7KHIROORZLQJ guidelines are recommended for determining when boosters should be adminis tered: $OOSHUVRQVZKRZRUNZLWKOLYHYLUXVLQDGLDJQRVWLFUHVHDUFKRUYDFFLQHSURGXF WLRQODERUDWRU\VKRXOGKDYHDVHUXPVDPSOHWHVWHGIRU91$HYHU\PRQWKVDQGD ERRVWHUYDFFLQDWLRQDGPLQLVWHUHGZKHQWKHWLWUHIDOOVEHORZ,8P/ WKLVLVWKH PLQLPXPUHFRPPHQGHGOHYHOLQGLFDWLYHRIDGHTXDWHLPPXQL]DWLRQ5HVSRQVLEOH DXWKRULWLHVVKRXOGHQVXUHWKDWDOOVWDIIDUHSURSHUO\LPPXQL]HG$OORWKHUSHUVRQV DWFRQWLQXLQJULVNRIH[SRVXUHWRO\VVDYLUXVHVVKRXOGKDYHDVHUXPVDPSOHWHVWHG IRU91$DERXWHYHU\\HDUV DERRVWHUYDFFLQDWLRQVKRXOGEHDGPLQLVWHUHGZKHQ WKHWLWUHIDOOVEHORZ Post-exposure prophylaxis All previously vaccinated staff exposed to a lyssavirus should receive local ZRXQGWUHDWPHQWDQGWZRERRVWHUGRVHVRIYDFFLQHRQGD\VDQG Biosafety Laboratory techniques in rabies Fifth edition Part 1. 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General considerations Chapter 4 The role of diagnostics in surveillance Introduction Comprehensive rabies prevention and control programmes are built on a IUDPHZRUNRIHIIHFWLYHVXUYHLOODQFHFDSDFLW\3XEOLFKHDOWKVXUYHLOODQFHFRQVLVWV of continuous systematic collection, analysis, interpretation and dissemina tion of information on health events (1, 2)7KHUHDUHPDQ\SRVVLEOHREMHFWLYHVRI surveillance: to demonstrate the presence and distribution of disease in humans and animals; to aid in control efforts; to determine the course of medical care for exposed individuals; or ultimately, to document absence of disease during elimination programmes (3)%\GHĺQLWLRQVXUYHLOODQFHLVDOZD\VOLQNHGWRVSHFLĺF DFWLRQV IRU PRQLWRULQJ LQWHUYHQWLRQV DQGRU LPPHGLDWH UHVSRQVHV )XUWKHUPRUH the capacity to detect, assess, notify and report on health events is a critical FRPSRQHQWRIWKH,QWHUQDWLRQDO+HDOWK5HJXODWLRQV (2)$GGLWLRQDOJXLGDQFH on rabies surveillance is available from WHO and OIE and in the rabies blue SULQW}(3–5) %CSGFGĂNKěKONS To promote awareness and vigilance, human and animal rabies must be noti ĺDEOHGLVHDVHVEDVHGRQVWDQGDUGFDVHGHĺQLWLRQV 7DEOH WKDWDUHSXEOLVKHG DQGZLGHO\GLVVHPLQDWHGE\QDWLRQDOKHDOWKDQGYHWHULQDU\VHUYLFHV6XUYHLOODQFH GDWDVKRXOGEHUHSRUWDEOHEDVHGRQWKHVHGHĺQLWLRQVWKURXJKDSSURSULDWHFKDQ QHOVE\SXEOLVKHGSURWRFROVWRIDFLOLWDWHWLPHO\GDWDVKDULQJDQGDQDO\VLV&DVH GHĺQLWLRQVVKRXOGEHIRUPXODWHGWRDOORZFODVVLĺFDWLRQEDVHGRQFOLQLFDOVLJQVRU V\PSWRPV VXVSHFWRUSUREDEOH RUGLDJQRVWLFWHVWV FRQĺUPHGRUQRQFDVH Methods of surveillance 0HDVXUHV RI LQFLGHQFH DUH HVVHQWLDO IRU UDELHV FRQWURO DQG SUHYHQWLRQ SURJUDPPHVWRHQVXUHDSSURSULDWHPDQDJHPHQWRIRXWEUHDNVWRPRQLWRUWUHQGV to evaluate the effectiveness of interventions and to estimate the burden of GLVHDVH'DWDFDQEHGHULYHGIURPQXPHURXVVXUYHLOODQFHPHWKRGVUDQJLQJIURP SXEOLF KHDOWK V\VWHPV WR URDGNLOO VXUYHLOODQFH WR GLUHFW VDPSOLQJ IURP DQLPDO SRSXODWLRQV (DFK PHWKRG KDV XQLTXH DSSOLFDWLRQV LQWHUSUHWDWLRQV DQG ODERUD WRU\WHVWLQJUHTXLUHPHQWVWKDWPXVWEHFRQVLGHUHG2IQRWHLVWKDWWHVWLQJKHDOWK\ DQLPDOVLVOLNHO\WR\LHOGQHJDWLYHUHVXOWVDQGLVWKHUHIRUHRIOLPLWHGYDOXHDVWKH SUHVHQFHRIUDELHVYLUXV 5$%9 FDQRQO\EHFRQĺUPHGLQWKHODWHRUĺQDOVWDJHVRI WKHGLVHDVHDQGWKHUHLVQRqFDUULHUVWDWHr VXEFOLQLFDOLQIHFWLRQ The role of diagnostics in surveillance Laboratory techniques in rabies Fifth edition Part 1. General considerations 7DEOH&DVHGHĺQLWLRQVIRUDQLPDODQGKXPDQUDELHV $QLPDOFDVHGHĺQLWLRQ +XPDQFDVHGHĺQLWLRQ Suspect case An animal that presents with any of A person presenting with an the following clinical signs: DFXWHQHXURORJLFDOV\QGURPH LH encephalitis) that progressively • hypersalivation worsens towards coma and death, W\SLFDOO\ZLWKLQtGD\VRIRQVHW • paralysis LIQRLQWHQVLYHFDUHLVLQVWLWXWHG0D\ • lethargy include any of the following signs and symptoms: sXQSURYRNHGDEQRUPDODJJUHVVLRQ ELWLQJłSHRSOHRUDQLPDOVDQGRU • aerophobia LQDQLPDWHREMHFWV • hydrophobia • abnormal vocalization • paraesthesia or localized pain • diurnal activity of nocturnal species • dysphagia sORFDOL]HGZHDNQHVV • nausea or vomiting Probable case A suspect animal with a history of a A suspect human with a reliable ELWHE\DQRWKHU VXVSHFWSUREDEOH KLVWRU\RIFRQWDFWZLWKD VXVSHFW FRQĺUPHG DQLPDO SUREDEOHFRQĺUPHG UDELGDQLPDO and/or $VXVSHFWDQLPDOWKDWLVNLOOHGGLHV RUGLVDSSHDUVZLWKLQtGD\VRI observing illness &RQĺUPHGFDVH A suspect or probable animal A suspect or probable human that FRQĺUPHGXVLQJDVWDQGDUG LVODERUDWRU\FRQĺUPHGXVLQJD GLDJQRVWLFWHVWDVGHĺQHGE\:+2 standard diagnostic test or OIE Not a case A suspect or probable animal A suspect or probable human that is in which rabies is ruled out by ruled out by appropriate laboratory appropriate laboratory testing testing or A suspect or probable human with no UHDVRQDEOHULVNRIDQLPDOFRQWDFWLQ WKHSDVWPRQWKV OIE, World Organisation for Animal Health; WHO, World Health Organization The role of diagnostics in surveillance Laboratory techniques in rabies Fifth edition Part 1. General considerations Passive public health surveillance Passive public health surveillance (PPHS) measures the incidence of disease at the human–animal interface, rather than the incidence within an animal popu ODWLRQ PRUHDFFXUDWHO\FDSWXUHGWKURXJKDFWLYHVXUYHLOODQFH 33+6LVSULPDULO\D public health intervention, of which the outcome of the intervention provides perti QHQWVXUYHLOODQFHGDWDIRUPRQLWRULQJDQGHYDOXDWLQJUDELHVFRQWUROSURJUDPPHV $}FRPSUHKHQVLYH33+6SURJUDPPHFRQVLVWVRIWKUHHFRUHDFWLYLWLHV L VXUYHLOODQFH IRUVXVSHFWHG5$%9H[SRVXUHV LHELWHHYHQWV LL VXUYHLOODQFHRIUDELHVVXVSHFW animals involved in human or domestic animal exposures; and (iii) monitoring of WKHXWLOL]DWLRQRISRVWH[SRVXUHSURSK\OD[LV 3(3 :KHQWKHVHWKUHHV\VWHPVDUH IRUPDOO\OLQNHGZLWKUHSRUWLQJDQGIHHGEDFNPHFKDQLVPVLWLVUHIHUUHGWRDVDQLQWH JUDWHGELWHFDVHPDQDJHPHQWSURJUDPPH6XFKSURJUDPPHVKDYHEHHQVKRZQ to reduce the unnecessary use of rabies PEP and increase adherence to rabies PEP schedules, emphasizing the importance of timely and accurate diagnosis (6) 7HVWUHVXOWVIURPUDELHVVXVSHFWDQLPDOVLGHQWLĺHGXQGHUD33+6SURJUDPPH are often used to determine the medical care provided to exposed humans and GRPHVWLFDQLPDOV$IDOVHtQHJDWLYHXQGHUWKHVHFRQGLWLRQVPD\UHVXOWLQWKHODFNRI SURYLVLRQRI3(3DQGDSRWHQWLDOIDWDORXWFRPHIRUWKHH[SRVHG8QGHUWKLVUXEULF WHVWVZLWKKLJKVSHFLĺFLW\DUHFULWLFDO,QIUHTXHQWIDOVHtSRVLWLYHVDUHQRWOLNHO\WR affect PPHS programmes negatively, and over application of PEP will rarely result LQDQHJDWLYHFOLQLFDORXWFRPH&XUUHQWO\:+2DQG2,(UHFRJQL]HWKH')$7 GLUHFW ĻXRUHVFHQWDQWLERG\WHVW WKH'5,7 GLUHFWUDSLGLPPXQRKLVWRFKHPLVWU\WHVW DQG UHYHUVHWUDQVFULSWDVHSRO\PHUDVHFKDLQUHDFWLRQ 573&5 DVSULPDU\GLDJQRVWLF WHVWVZKHQGHFLVLRQVRQ3(3PD\EHDIIHFWHG 7DEOH} $OWHUQDWLYHWHVWVVXFK DV ODWHUDO ĻRZ DVVD\V VKRXOG QRW EH XVHG DV D VROH GLDJQRVWLF DVVD\ LQ WKH context of a passive surveillance programme until they have been fully evaluated DQGUHFRJQL]HGE\WKHUHOHYDQWLQWHUQDWLRQDORUJDQL]DWLRQV5RXWLQHPRQLWRULQJRI GLDJQRVWLFSURĺFLHQF\RIODERUDWRULDQVVKRXOGEHFRQGXFWHGDQGLQVLWXDWLRQV ZKHUHSURĺFLHQF\FDQQRWEHJXDUDQWHHGRUFRQĺUPDWLRQRISULPDU\GLDJQRVWLFWHVW UHVXOWVLVQHHGHGFRQĺUPDWRU\WHVWLQJE\DQRWKHUPHWKRG HJ}')$7FRQĺUPHG E\3&5 Consideration of sample quality is more important within the context of a PPHS SURJUDPPHWKDQLQDFWLYHVXUYHLOODQFHSURJUDPPHV7KHQHXURWURSLFSDWKZD\V WUDYHUVHGE\5$%9PD\OHDGWRLQIHFWLRQLQRQO\DSRUWLRQRIWKHEUDLQVWHP WKHUH IRUH WHVWLQJ D IXOO FURVVVHFWLRQ LV UHTXLUHG WR GHĺQLWLYHO\ UXOH RXW 5$%9 LQIHF WLRQ}(4):KHQDIXOOFURVVVHFWLRQRIEUDLQVWHPFDQQRWEHREWDLQHGRUWKHVDPSOH TXDOLW\LVLQVXIĺFLHQWWRHQDEOHLGHQWLĺFDWLRQRIDQDWRPLFVWUXFWXUHVDQG5$%9LV QRWLGHQWLĺHGE\WKH')$7WKH'5,7RU573&5DQGFRQĺUPDWRU\WHVWVDUHQRW DYDLODEOHWKHVDPSOHVKRXOGEHGHFODUHGLQGHWHUPLQDWH,QWKLVVLWXDWLRQSXEOLF KHDOWKRIĺFLDOVVKRXOGIRUPXODWH3(3GHFLVLRQVEDVHGRQDFRPSOHWHHYDOXDWLRQRI the circumstances of the bite event, local rabies epidemiology and an assessment RIWKHRIIHQGLQJDQLPDO 5HSRUWLQJDQGQRWLĺFDWLRQSURWRFROVDUHLPSRUWDQWFRQVLGHUDWLRQVLQWKHFRQWH[W RID33+6SURJUDPPH/DERUDWRULHVVKRXOGEHIDPLOLDUZLWKPHFKDQLVPVIRUWUDQV SRUWDWLRQDQGVXEPLVVLRQRIVDPSOHVDQGEHDEOHWRUDSLGO\WHVWDQGUHSRUWUHVXOWV 6DPSOHVVKRXOGEHWHVWHGUDSLGO\DQGUHSRUWHGQROHVVWKDQZLWKLQtGD\VRIWKH exposure event to ensure that the results can be used to inform PEP decisions, if The role of diagnostics in surveillance Laboratory techniques in rabies Fifth edition Part 1. General considerations 7DEOH6HOHFWLYHFRPSDULVRQRIYDULRXVUDELHVWHVWVDQGWKHLUXWLOLW\LQGLIIHUHQWVXUYHLOODQFHSURJUDPPHV Test Passive Active Clinical Sample types Notes surveillance surveillance diagnosis 5HYHUVH %UDLQ Used as a primary transcriptase 6NLQELRSV\ diagnostic assay with polymerase chain sequencing and as a Saliva reaction FRQĺUPDWRU\WHVW 573&5 +HPLQHVWHG %UDLQ Used as a primary 573&5 6NLQELRSV\ diagnostic assay with sequencing and as a Saliva FRQĺUPDWRU\WHVW 5HDOWLPH %UDLQ Used as a primary 0ROHFXODU 573&5 6NLQELRSV\ diagnostic assay Saliva 'LUHFWĻXRUHVFHQW %UDLQ s3ULPDU\qJROG antibody test 6NLQELRSV\ standard” test ')$7 s5HFRJQL]HGE\2,( and WHO Direct rapid %UDLQ • Primary test LPPXQR 6NLQELRSV\ histochemistry s5HFRJQL]HGE\2,( WHVW '5,7 and WHO Indirect rapid %UDLQ Can also be used for LPPXQR 6NLQELRSV\ antigenic typing studies histochemistry WHVW ,5,7 Antigen ,PPXQR %UDLQ s3RWHQWLDOO\ORZFRVW chromatographic WHVW LHODWHUDO • Transportable ĻRZGHYLFHV s9DULDEOHVHQVLWLYLW\DQG VSHFLĺFLW\ 5DSLGĻXRUHVFHQW Serum &DQEHXVHIXOLQGHĺQLQJ focus inhibition &6) disease activity as part WHVW 5)),7 of surveys in new areas )OXRUHVFHQW and species (such as antibody virus bats) neutralization test )$91 Indirect Serum 5HTXLUHVVXLWDEOHFRQWURO ĻXRUHVFHQW &6) materials DQWLERG\WHVW ,)$ Antibody Indirect Serum HQ]\PHOLQNHG immunosorbent assay (ELISA) Competitive Serum 0D\EHXVHIXOLQ ELISA immunogenicity studies, such as evaluation of wildlife vaccination programmes &6)FHUHEURVSLQDOĻXLG 2,(:RUOG2UJDQLVDWLRQIRU$QLPDO+HDOWK :+2:RUOG+HDOWK2UJDQL]DWLRQ tQRWUHFRPPHQGHG ORZUHFRPPHQGDWLRQXVHIXOIRUFRQĺUPDWRU\WHVWLQJ PRGHUDWHUHFRPPHQGDWLRQXVHIXOIRUSULPDU\RU FRQĺUPDWRU\WHVWLQJ KLJKUHFRPPHQGDWLRQXVHIXOIRUSULPDU\RUFRQĺUPDWRU\WHVWLQJ The role of diagnostics in surveillance Laboratory techniques in rabies Fifth edition Part 1. General considerations WKH\KDYHQRWDOUHDG\EHJXQ6WDQGDUGUHSRUWLQJSURWRFROVVKRXOGEHLQSODFHVR that results are integrated with regional and national reporting structures to ensure UHVXOWVDUHDOVRLQFOXGHGLQHSLGHPLRORJLFDODQDO\VLVDQGUHSRUWV7KHGHFLVLRQWR LQLWLDWH3(3VKRXOGEHGHWHUPLQHGE\SXEOLFKHDOWKRIĺFLDOVZLWKNQRZOHGJHRIWKH circumstances of the exposure event, in accordance with WHO recommenda WLRQVDQGVKRXOGQRWEHVROHO\GHSHQGHQWRQODERUDWRU\WHVWLQJUHVXOWV Active or enhanced surveillance Active surveillance (sometimes referred to as enhanced surveillance) is the WDUJHWHGVDPSOLQJRIDQLPDOVLQDSRSXODWLRQIRUGHWHUPLQLQJGLVHDVHLQFLGHQFH This method of surveillance is a more accurate measure of the true occurrence of rabies in the targeted population, but may still be biased depending on the choice RIVDPSOLQJWHFKQLTXH$FWLYHVXUYHLOODQFHGRHVQRWLQFOXGHWKHLQYHVWLJDWLRQRI UDELHVVXVSHFW DQLPDOV LQYROYHG LQ KXPDQ RU DQLPDO H[SRVXUH HYHQWV SDVVLYH SXEOLF KHDOWK VXUYHLOODQFH $FWLYH VXUYHLOODQFH LV W\SLFDOO\ HQDFWHG WR DQVZHU VSHFLĺFTXHVWLRQVUHODWHGWRDQLQWHUYHQWLRQRUWRLPSURYHVLWXDWLRQDODZDUHQHVV IRUPDNLQJUDELHVFRQWUROGHFLVLRQV 6WXGLHVKDYHVKRZQWKDW5$%9KDVDUHODWLYHO\ORZUHSURGXFWLYHQXPEHUDQG a long incubation period (7, 8) 7KHUHIRUH LQ DQ HQGHPLF VWDWH RI WUDQVPLVVLRQ LQGLVFULPLQDWHVDPSOLQJRIDSRSXODWLRQLVOLNHO\WRUHVXOWLQDORZUDWHRIGHWHFWLRQ :KLOHDFWLYHVXUYHLOODQFHLVDEHWWHUUHĻHFWLRQRIWKHWUXHWUDQVPLVVLRQG\QDPLFV LQDSRSXODWLRQLQGLVFULPLQDWHWHVWLQJRIKHDOWK\VXEMHFWVUHTXLUHVKLJKOHYHOVRI WHUPLQDO VDPSOLQJ WKDW PD\ QRW EH HFRQRPLFDOO\ RU HWKLFDOO\ IHDVLEOH 7DUJHWHG DFWLYH VXUYHLOODQFH HJ QXLVDQFH DQLPDOV URDG NLOO PD\ SURYLGH D KLJKHU UDWH of detection; however, the resulting rates will be less representative of the entire population of interest (9)$FWLYHVXUYHLOODQFHLVRIWHQLPSOHPHQWHGLQFRQMXQFWLRQ with a PPHS programme to provide a comprehensive understanding of the rabies epidemiology in the target area (10) Laboratory diagnostics are an essential component of an active surveillance SURJUDPPH7KHLPSRUWDQFHRILQFUHDVHGVHQVLWLYLW\DQGVSHFLĺFLW\RIWKHWHVWV XVHG IRU DFWLYH VXUYHLOODQFH VKRXOG EH EDODQFHG EDVHG RQ SURJUDPPH REMHF WLYHV:KLOHFOLQLFDORXWFRPHVRISHUVRQVLQYROYHGLQDQH[SRVXUHVKRXOGQRWEH a concern for these programmes, misdiagnosis may have substantial impacts RQPDQDJHPHQWSUDFWLFHVDQGGHWHUPLQDWLRQRIDGLVHDVHIUHHVWDWXV7KHUHIRUH SULPDU\GLDJQRVWLFPHWKRGV HJ')$7'5,7573&5 UHFRJQL]HGE\2,(RU:+2 VKRXOGEHSULRULWL]HG 7DEOH +RZHYHULQWKHFRQWH[WRIDQDFWLYHVXUYHLOODQFH SURJUDPPHVHQVLWLYLW\DQGVSHFLĺFLW\PD\EHVDFULĺFHGDWWLPHVZKHQSULPDU\ PHWKRGVDUHQRWDYDLODEOHRUHFRQRPLFDOO\IHDVLEOH 1RQSULPDU\GLDJQRVWLFPHWKRGVVXFKDVODWHUDOĻRZDVVD\V /)$ KDYHVKRZQ variable reliability between manufacturers and manufacturing lots; however, these SURGXFWVDUHHDVLO\WUDQVSRUWDEOHDQGSURYLGHDĺHOGUHDG\GLDJQRVWLFRSWLRQ(11, 12)$OVRQXPHURXVPROHFXODUDVVD\VIRUGHWHFWLRQRI5$%9KDYHEHHQGHVFULEHG DVRXWOLQHGLQWKLVPDQXDO KRZHYHUWKHLUVHQVLWLYLW\DQGVSHFLĺFLW\FDQGLIIHUE\ WHFKQLTXH DQG FURVVFRQWDPLQDWLRQ PD\ UHVXOW LQ IDOVHtSRVLWLYH UHVXOWV ZKHQ XVHGXQGHUĺHOGFRQGLWLRQV(13, 14)$FWLYHVXUYHLOODQFHSURJUDPPHVWKDWFKRRVH WRXVHQRQSULPDU\GLDJQRVWLFDVVD\VVKRXOGFRQVLGHUSHULRGLFYDOLGDWLRQRIWKHLU UHVXOWVEHIRUHPDNLQJVXEVWDQWLDOSURJUDPPDWLFFKDQJHV$YDOLGDWLRQVXEVHWRI The role of diagnostics in surveillance Laboratory techniques in rabies Fifth edition Part 1. 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General considerations Chapter 5 An overview of antemortem and postmortem tests for diagnosis of human rabies Introduction 5DELHVRQHRIWKHPRVWQHJOHFWHG]RRQRWLFGLVHDVHVZRUOGZLGHGLVSURSRUWLR QDWHO\DIIHFWVFKLOGUHQDQGLPSRYHULVKHGUXUDOFRPPXQLWLHV7KHWUXHEXUGHQRI the disease and its public health impact remain underestimated and can be attri EXWHGWRLQDGHTXDWHRUQRQH[LVWHQWVXUYHLOODQFHRIDQLPDODQGKXPDQUDELHVODFN of laboratory diagnostic facilities, misdiagnosis of rabies, underreporting of cases E\ORFDODQGFHQWUDODXWKRULWLHVDQGLQDGHTXDWHOHJLVODWLRQIRUFRPSXOVRU\QRWLĺ FDWLRQRIFDVHV7KHODFNRIDFFXUDWHGDWDRQGLVHDVHEXUGHQXSRQZKLFKUHJLRQDO and national priorities for research and control are based, further perpetuates a cycle of indifference and neglect (1),WLVHVVHQWLDOWKHUHIRUHWRLPSURYHHSLGH miological surveillance using diagnostic approaches that are based on validated SURWRFROVDQGVSHFLPHQVDQGHYDOXDWHGXQGHUĺHOGFRQGLWLRQV&OLQLFDOGLDJQRVLV RIKXPDQUDELHVLVFKDOOHQJLQJDQGRIWHQXQUHOLDEOH/DERUDWRU\FRQĺUPDWLRQPXVW therefore be done in all suspected cases wherever feasible (2) 6LJQLĺFDQW SURJUHVV KDV EHHQ PDGH LQ UDELHV ODERUDWRU\ WHFKQLTXHV GXULQJ WKH SDVW IHZ GHFDGHV HVSHFLDOO\ IRU DQWHPRUWHP GLDJQRVLV 6HYHUDO YDOLGDWHG WHFKQLTXHVDUHQRZDYDLODEOHWRGHWHFWVSHFLĺFYLUDODQWLJHQVQXFOHLFDFLGVRU DQWLERGLHVLQVHOHFWHGELRORJLFDOVDPSOHV7KHVHOHFWLRQFROOHFWLRQDQGVKLSPHQW of biological specimens are crucial steps, particularly for antemortem diagnosis, VLQFH VHYHUDO IDFWRUV VXFK DV WKH GXUDWLRQ RI LOOQHVV SULRU YDFFLQDWLRQ VLQJOH PXOWLSOHVHULDOFOLQLFDOVDPSOHVWHVWHGTXDOLW\RIVDPSOHVDQGW\SHRIDVVD\XVHG LQĻXHQFHWKHWHVWUHVXOWV)XUWKHUPRUHWKHFKRLFHRIGLDJQRVWLFWHVWVDOVRODUJHO\ depends on the availability of infrastructure and technical expertise, and may vary LQVHWWLQJVLQZKLFKUDELHVLVHQGHPLFRUQRQHQGHPLF(3) This chapter describes the various types of assays and clinical samples that FDQEHXVHGIRUDQWHPRUWHPDQGSRVWPRUWHPGLDJQRVLVRIKXPDQUDELHV7KHDLP LVWRVHUYHDVDQLQWURGXFWRU\JXLGHEHIRUHUHIHUHQFHWRVSHFLĺFGLDJQRVWLFDVVD\V GHWDLOHGHOVHZKHUHLQWKLVPDQXDO&OHDUO\WKHSHUIRUPDQFHRIHDFKODERUDWRU\LV KLJKO\GHSHQGHQWRQTXDOLW\DVVXUDQFHDQGTXDOLW\FRQWUROSUDFWLFHV$OOODERUD tories are encouraged to develop and implement stringent quality management VWDQGDUGV LQFOXGLQJ SHULRGLF SDUWLFLSDWLRQ LQ LQWHUQDO DQG H[WHUQDO SURĺFLHQF\ testing programmes (2) Antemortem and postmortem tests for diagnosis of human rabies Laboratory techniques in rabies Fifth edition Part 1. General considerations .CDORCěORXEONĂRMCěKONěORTĚGOTěEĚKNKECĚ mimics 7ZR PDMRU DQG GLVWLQFW FOLQLFDO IRUPV RI UDELHV KDYH EHHQ UHFRJQL]HG LQ humans: classical encephalitic (furious) rabies and paralytic rabies (4)%DVHGRQ WKHVHGHVFULSWLRQVWKHVWDQGDUGFOLQLFDOFDVHGHĺQLWLRQFRUUHVSRQGVWRDSDWLHQW SUHVHQWLQJZLWKDQDFXWHQHXURORJLFDOV\QGURPH LHHQFHSKDOLWLV GRPLQDWHGE\ forms of hyperactivity or paralytic syndromes progressing towards coma and GHDWKXVXDOO\E\FDUGLDFRUUHVSLUDWRU\IDLOXUHDQGW\SLFDOO\ZLWKLQtGD\VDIWHU WKHĺUVWVLJQLIQRLQWHQVLYHFDUHLVLQVWLWXWHG(2) However, a diagnosis of rabies based solely on clinical features, especially in WKHDEVHQFHRIDKLVWRU\RIH[SRVXUHLVGLIĺFXOWDQGRIWHQXQUHOLDEOH(2)/DERUDWRU\ FRQĺUPDWLRQPXVWEHGRQHZKHUHYHUIHDVLEOH,QGHHGWKHFOLQLFDOSUHVHQWDWLRQRI rabies is often variable and may represent a continuum of signs and symptoms (5),QDGGLWLRQDW\SLFDODQGQRQFODVVLFDOFOLQLFDOIRUPVRIUDELHVDUHLQFUHDVLQJO\ being recognized (2, 4–7) )XUWKHUPRUH UDELHV FDQ EH FRQIXVHG FOLQLFDOO\ ZLWK *XLOODLQt%DUUÆV\QGURPH RIWHQFOLQLFDOO\LQGLVWLQJXLVKDEOHIURPWKHSDUDO\WLFIRUP of rabies) or even with other infectious etiologies, such as cerebral malaria or KHUSHV VLPSOH[ HQFHSKDOLWLV 3V\FKLDWULF GLVRUGHUV SRVWYDFFLQDO HQFHSKDOLWLV DQWL10'$ UHFHSWRU HQFHSKDOLWLV VFRUSLRQ DQG VQDNH HQYHQRPDWLRQV LOOLFLW drug use and organophosphate poisoning all can also cause a clinical syndrome suggestive of rabies (5)5HSRUWVRIUDELHVEHLQJWUDQVPLWWHGWKURXJKWLVVXHDQG organ transplants (8, 9) also emphasize the need for scrupulous organ donor screening for rabies, especially in donors with a neurological illness of unexplained HWLRORJ\DQGWKHSUHVHQFHRIRWKHUULVNIDFWRUVIRUUDELHV Clinical management and public health measures Early diagnosis can obviate the need for unwarranted medical tests and treat ment, and help also in prognostication, case closure and grief counselling with IDPLO\PHPEHUV$QWHPRUWHPFRQĺUPDWLRQRIGLDJQRVLVLVFUXFLDOWRDLGSK\VL cians contemplating imminent treatment (critical care or experimental therapeutic RSWLRQV RUSDOOLDWLRQ/DERUDWRU\WHVWVVXFKDVYLUDOORDGDQGQHXWUDOL]LQJDQWLERG\ WLWUHVPD\DOVREHXVHIXOWRPRQLWRUWKHFOLQLFDOFRXUVHRILOOQHVVDQGWKHSDWLHQWpV response to therapy (2, 5) /DERUDWRU\FRQĺUPDWLRQRIUDELHVLQDKRVSLWDOL]HGSDWLHQWDOVRDLGVLQLQVWLWX ting appropriate infection control measures, including barrier nursing and admi QLVWUDWLRQRISRVWH[SRVXUHSURSK\OD[LV 3(3 WRKHDOWKFDUHZRUNHUVDQGIDPLO\ PHPEHUV H[SRVHG WR WKH SDWLHQWpV LQIHFWLRXV VHFUHWLRQV (2) /DERUDWRU\ FRQĺU mation is imperative for clinical management as well as to guide public health PHDVXUHVHVSHFLDOO\LQQRQHQGHPLFVHWWLQJVWRGHWHFWLPSRUWDWLRQRUHPHUJHQFH of rabies and to institute PEP to others who may have been potentially exposed to WKHVDPHVRXUFH/DERUDWRU\WHVWVDOVRDLGLQGRFXPHQWLQJFRQWURODQGUHVHDUFK HJVHURSUHYDOHQFHVWXGLHV3(3IDLOXUHV Antemortem and postmortem tests for diagnosis of human rabies Laboratory techniques in rabies Fifth edition Part 1. General considerations Disease surveillance The true human burden of this neglected zoonotic disease remains underes timated, particularly in rabies endemic countries in Asia and Africa due to limited UHSRUWLQJRIODERUDWRU\FRQĺUPHGFDVHV5HJXODUUHSRUWLQJRIGLDJQRVWLFGDWDLV crucial for estimation of disease burden and helps prioritize resources towards prevention of human rabies (10)&RQWLQXDOODERUDWRU\EDVHGVXUYHLOODQFHLVHVVHQ WLDOHYHQLQSUHYLRXVO\qUDELHVIUHHrFRXQWULHV$VORQJDVIRFLRIZLOGOLIHRUFDQLQH rabies exist anywhere and transborder migration of infected animals from enzootic DUHDVRULQWHUQDWLRQDOWUDYHODQGJOREDOWUDGHRIOLYHVWRFNSHWVDQGZLOGOLIHFRQWLQXH WKHWKUHDWRIUHLQWURGXFWLRQRIUDELHVDOZD\VH[LVWV$GGLWLRQDOO\VRPHODERUDWRU\ tests can aid in characterization of the virus as well as in molecular epidemiolo gical studies to monitor the disease dynamics at regional, national and interna tional levels, and identify the source of emergence of the disease in previously UDELHVIUHHDUHDV(1, 11) Antemortem diagnostic tests 6HYHUDOODERUDWRU\WHFKQLTXHVDUHDYDLODEOHIRUDQWHPRUWHPFRQĺUPDWLRQRID VXVSHFWHGRUSUREDEOHFDVHRIUDELHV 7DEOH 7HFKQLTXHVIRUYLUDOQXFOHLFDFLG GHWHFWLRQVXFKDVUHYHUVHWUDQVFULSWDVHDQGSRO\PHUDVHFKDLQUHDFWLRQ 573&5 RURWKHUQXFOHLFDFLGDPSOLĺFDWLRQDQGGHWHFWLRQPHWKRGV HJ1$6%$>QXFOHLF DFLG VHTXHQFH EDVHG DPSOLĺFDWLRQ@ XVLQJ FOLQLFDO VDPSOHV VXFK DV VDOLYD QXFKDO VNLQ H[WUDFWHG KDLU IROOLFOHV WHDUV XULQH DQG FHUHEURVSLQDO ĻXLG &6) KDYHDFKLHYHGDUDSLGUHGXFWLRQLQWXUQDURXQGWLPHV7KHVHWHVWVDUHLQURXWLQH use in several centralized and reference laboratories globally, especially for ante mortem diagnosis, since traditional methods are fraught with several limitations (10, 12)6XFKWHVWLQJSHUPLWVDQWHPRUWHPFRQĺUPDWLRQRIUDELHVLQFOLQLFDOVDPSOHV obtained by less invasive techniques and can therefore alleviate the requirement for autopsy or postmortem brain biopsy, and the accompanying logistics and safety procedures (13–15) The sensitivity of these techniques for antemortem diagnosis varies according to the clinical form of rabies, stage of the disease, immunological status of host, LQWHUPLWWHQWYLUDOH[FUHWLRQDQGWKHWHFKQLFDOH[SHUWLVHRIWKHODERUDWRU\+RZHYHU VWXGLHVKDYHGHPRQVWUDWHGWKDWWHVWLQJDWOHDVWWKUHHVDPSOHVRIVDOLYDWDNHQDW tKLQWHUYDOVWRJHWKHUZLWKDQXFKDOVNLQELRSV\DSSURDFKHVVHQVLWLYLW\ in the encephalitic form of rabies (15)2WKHUVDPSOHVVXFKDV&6)DQGXULQHKDYH PRUHOLPLWHGVHQVLWLYLW\7HDUVKDYHEHHQSURSRVHGEXWKDYHQRWEHHQYDOLGDWHG RQDODUJHVFDOH$VZLWKDQ\RWKHUGLDJQRVWLFDVVD\VSURFHGXUHVIRUVWULQJHQW YDOLGDWLRQDQGTXDOLW\FRQWURODUHLPSHUDWLYH:KLOHDSRVLWLYHYDOLGDWHGUHVXOWLV indicative of rabies, a negative result does not essentially rule out a diagnosis RIUDELHVLQDOOFDVHVZKLFKLVDPDMRUOLPLWDWLRQRIDQWHPRUWHPWHVWLQJ(2)$QWH mortem brain biopsy is not recommended as a primary test, as it is impractical and false–negative results can be obtained due to sampling error especially in the early stages of illness (16) 6HURORJLFDO WHVWV VXFK DV WKH UDSLG ĻXRUHVFHQW IRFXV LQKLELWLRQ WHVW 5)),7 DQGWKHĻXRUHVFHQWDQWLERG\YLUXVQHXWUDOL]DWLRQWHVW )$91 ZKLFKGHWHFWQHXWUD OL]LQJDQWLERGLHVDQG(/,6$IRUGHWHFWLRQRIVSHFLĺFDQWLUDELHVDQWLERGLHVKDYH Antemortem and postmortem tests for diagnosis of human rabies Laboratory techniques in rabies Fifth edition Part 1. General considerations 7DEOH+XPDQUDELHVDQWHPRUWHPGLDJQRVWLFWHVWV Test Target Clinical Advantages Limitations Additional comments samples Nucleic acid 9LUDO51$ Saliva (DV\QRQLQYDVLYH 6HULDOSRROHG 5DSLGWXUQDURXQGWLPH DPSOLĺFDWLRQ sample collection; samples need tK DPSOLFRQVIURP (NAA) high sensitivity to be tested FRQYHQWLRQDO QRWUHDO for increased WLPH 3&5FDQEHXVHGIRU Conventional sensitivity due to genetic characterization DQGUHDO intermittent virus time reverse shedding (at least 5LVNRIDPSOLFRQ transcriptase VHULDOVDPSOHV contamination leading polymerase DWtKLQWHUYDOV to false– positive results chain reaction KLJKHULQFRQYHQWLRQDO3&5 1XFKDOVNLQ High sensitivity in 5HTXLUHVIXOO 573&5 DV ELRSV\KDLU all stages of illness WKLFNQHVVELRSV\ well as other Quality of clinical follicles and adequate methods specimens vital; hair follicles for appropriate storage and increased sensi transport conditions must tivity be maintained &6) Sample usually Invasive tech available in the nique for sample 5HTXLUHPHQWIRU context of ence collection, low infrastructure and stringent phalitis sensitivity quality control Can also be performed on other clinical samples such as urine and tears, but sensitivity low 5DSLG 9LUXV &6) Diagnostic of Low sensitivity, Sensitivity of test rises ĻXRUHVFHQW neutralizing rabies irrespective late appearance with increased duration of focus inhibition antibodies of previous RIVSHFLĺF survival WHVW 5)),7 vaccination status antibodies )OXRUHVFHQW 5)),7 !}GD\V VKRUW )RU5)),7DQG)$91 antibody virus )$91 RU 9DOXDEOHWRPRQLWRU survival and late ORQJWXUQDURXQGWLPH t neutralization antibodies the immune seroconversion days); requirement for cell WHVW )$91 against response within in rabies limit FXOWXUHIDFLOLW\ĻXRUHVFHQFH rabies the central nervous diagnostic value microscope and biosafety (Q]\PHOLQNHG glycoprotein system and thus a measures; test procedure immunosorbent (ELISA) possible clearance Titre in serum cumbersome assay (ELISA) of the rabies must be virus in patients interpreted 7HVW 5)),7)$91 LV being treated cautiously in sensitive to cytotoxicity with experimental previously LQSRRUTXDOLW\VHUD protocols vaccinated QRQVSHFLĺFLQKLELWRUVRI individuals; virus in sera may produce VLJQLĺFDQWULVHLQ false– positive results paired sera to be Adequately validated ELISA demonstrated for for antibody detection can FRQĺUPDWLRQRI %ORRG Diagnostic of EHXVHGLI5)),7RU)$91 diagnosis (serum) rabies in individuals is unavailable; these tests not vaccinated are rapid, inexpensive and previously safe since live virus is not required 9DOXDEOHLQ HVWLPDWLRQRISRVW Detection of antibodies to vaccinal antibody RWKHUYLUDODQWLJHQV HJ titres nucleoprotein) may also be useful, as they may appear before neutralizing antibodies Antemortem and postmortem tests for diagnosis of human rabies Laboratory techniques in rabies Fifth edition Part 1. General considerations 7DEOH&RQWLQXHG Test Target Clinical Advantages Limitations Additional comments samples Direct 9LUDODQWLJHQ Corneal Easy and rapid Low sensitivity 5HTXLUHPHQWIRUH[SHQVLYH ĻXRUHVFHQW smear technique DQGVSHFLĺFLW\ ĻXRUHVFHQFHPLFURVFRSH antibody test ')$7 Not Operator expertise required recommended for interpretation because of ULVNRIFRUQHDO Preferable to use more VFDULĺFDWLRQ VHQVLWLYHYLUDO51$ detection methods (such as 573&5 RQVNLQVDPSOHV Nuchal 0RGHUDWHVHQVLWLYLW\ Cumbersome, VNLQ IUR]HQ at all stages WLPHFRQVXPLQJ section) of illness; can technique; also be used for requires multiple postmortem rabies sections with hair diagnosis if brain follicles to be tissue unavailable examined Cryostat required to prepare frozen VHFWLRQVRIVNLQt not practicable in all settings 5DELHVWLVVXH Live virus %LRORJLFDO Enables Low sensitivity, Use as potential culture infection ĻXLGV &6) propagation of longer turnaround FRPSOHPHQWDU\WHVW WHVW 57&,7 saliva) virus and valuable WLPH t}GD\V for obtaining large requires Not feasible for use as quantities of virus ĻXRUHVFHQFH routine diagnostic test; for molecular microscope, can be done only in characterization cell culture specialized laboratories for and other studies and adequate FRQĺUPDWRU\GLDJQRVLVDQG biocontainment for further characterization facilities, of viruses and operator expertise, requires infectious virus in the sample &6)FHUHEURVSLQDOĻXLG 51$ULERQXFOHLFDFLG a limited role in diagnosis of rabies, due to the rapid progression of illness and VKRUW}VXUYLYDO+RZHYHUGHWHFWLRQRIVSHFLĺFDQWLUDELHVYLUXVDQWLERGLHVLQVHUXP LQ}XQYDFFLQDWHGLQGLYLGXDOV DQG&6)LVYDOXDEOHIRUGLDJQRVLVLQFDVHVHVSHFLDOO\ ZKHUHVXUYLYDOLVSURORQJHGEH\RQGDZHHN(7, 14)0RUHRYHUDQLQYHUVHFRUUHODWLRQ KDVEHHQREVHUYHGEHWZHHQWKHSUHVHQFHRIDQWLUDELHVYLUXVDQWLERGLHVLQ&6) DQGWKHGHWHFWLRQRIYLUDO51$LQFOLQLFDOVDPSOHV(14, 17)7KHUHIRUHDFRPELQD tion of several tests conducted on multiple clinical samples, with serial sampling whenever feasible (because of intermittent virus shedding in saliva, and a poten WLDOULVHLQDQWLERG\WLWUHVLQVHULDO&6)DQGVHUXPVDPSOHV LVUHFRPPHQGHGWR increase the sensitivity of antemortem diagnosis (7, 10)$VXJJHVWHGDOJRULWKPIRU DQWHPRUWHPGLDJQRVLVLVLOOXVWUDWHGLQ)LJ Antemortem and postmortem tests for diagnosis of human rabies Laboratory techniques in rabies Fifth edition Part 1. General considerations ANTEMORTEM Anti-rabies antibodies Viral RNA detection (RT-PCR)a,b RFFIT/FAVN/ELISA Salivac Nuchal CSF CSF Serum skin/Hair follicles Positive Negative Positive Diagnosis of Rabies cannot rabies if NEVER be ruled out vaccinated Negative Positive in single or in all samples multiple samples Test another If previously sample after vaccinated look 7–10 days for rise in titres Rabies cannot &RQĺUPHG d be ruled out rabies Positive Negative &RQĺUPHG Unlikely to be rabies; rabies but cannot be ruled out &6)FHUHEURVSLQDOĻXLG (/,6$HQ]\PHOLQNHGLPPXQRVRUEHQWDVVD\ )$91ĻXRUHVFHQWDQWLERG\YLUXVQHXWUDOL]DWLRQ WHVW 5)),7UDSLGĻXRUHVFHQWIRFXVLQKLELWLRQWHVW 57&,7UDELHVWLVVXHFXOWXUHLQIHFWLRQWHVW 573&5 UHYHUVH transcriptase polymerase chain reaction a$OWHUQDWLYH DOEHLWOHVVVHQVLWLYH WHFKQLTXHVLQFOXGHDQWLJHQGHWHFWLRQE\')$7LQQXFKDOVNLQDQGYLUXVLVRODWLRQE\ 57&,7LQVDOLYDDQG&6)1HJDWLYHWHVWUHVXOWVFDQQRWUXOHRXWUDELHV b Other clinical samples include tears and urine; however, tests on these samples are less sensitive and have not been YDOLGDWHGRQODUJHVDPSOHVL]HV c7HVWLQJRQDWOHDVWWKUHHVHULDOVDOLYDVDPSOHVFROOHFWHGDWtKLQWHUYDOVLVUHFRPPHQGHGGXHWRLQWHUPLWWHQWYLUDO VKHGGLQJ d7HVWLQJWKUHHVHULDOVDPSOHVWRJHWKHUZLWKDQXFKDOVNLQELRSV\DSSURDFKHVVHQVLWLYLW\LQWKHHQFHSKDOLWLFIRUP RIUDELHVEXWQHJDWLYHUHVXOWVFDQQRWUXOHRXWUDELHVLQSDUDO\WLFRURWKHUDW\SLFDOIRUPV )LJ$OJRULWKPIRUKXPDQUDELHVDQWHPRUWHPGLDJQRVLV Antemortem and postmortem tests for diagnosis of human rabies Laboratory techniques in rabies Fifth edition Part 1. General considerations Postmortem diagnostic tests Demonstration of rabies virus nucleoprotein antigens in brain tissue by the GLUHFWĻXRUHVFHQWDQWLERG\WHVW ')$7 LVWKHPRVWZLGHO\XVHGWHVWIRUSRVWPRUWHP rabies diagnosis, when brain tissue is available for testing (see Chapter 11 7KH}GLUHFWUDSLGLPPXQRKLVWRFKHPLVWU\WHVW '5,7 IRUGHWHFWLRQRIYLUDODQWLJHQV LQEUDLQWLVVXHKDVEHHQIRXQGWREHFRPSDUDEOHWRWKH')$7LQWKHGLDJQRVLVRI DQLPDOUDELHVDQGKDVWKHDGGLWLRQDODGYDQWDJHRIDSSOLFDELOLW\XQGHUĺHOGFRQGL WLRQVDVDQH[SHQVLYHĻXRUHVFHQFHPLFURVFRSHLVQRWUHTXLUHGIRULQWHUSUHWDWLRQ of results (see Chapter 12) (18) :KLOH WKHUH LV D QHHG IRU DGHTXDWH YDOLGDWLRQ RIWKLVWHVWVSHFLĺFDOO\IRUGLDJQRVLVRIKXPDQUDELHVLWLVQRWH[SHFWHGWRYDU\ as compared to the tens of thousands of tests conducted upon multiple other PDPPDOLDQVSHFLHV2WKHUWHVWVIRUSRVWPRUWHPGLDJQRVLVDQGWKHLUDGYDQWDJHV DQGOLPLWDWLRQVDUHVXPPDUL]HGLQ7DEOH Postmortem brain tissue can be obtained by craniotomy; however, this proce dure continues to be a challenge due to declining rates of autopsies related to UHOLJLRXVFXOWXUDODQGRWKHUIDFWRUV%UDLQELRSV\LVWKHUHIRUHWKHSUHIHUUHGSRVW mortem sampling technique which can be obtained via the orbital or transnasal route using biopsy needles or through the occipital route through the foramen PDJQXP XVLQJ OXPEDU SXQFWXUH QHHGOHV 7KH WUDQVQDVDO RU RUELWDO URXWH ZLOO contain fragments of orbitofrontal cortex, whereas the transforamen magnum route will obtain samples from the cerebellum and brain stem (16, 19–22) :KHUHYHU EUDLQ WLVVXH LV QRW DYDLODEOH IRU FRQĺUPDWLRQ RI D GLDJQRVLV SRVW PRUWHP ZKLFK LV RIWHQ WKH FDVH WHVWLQJ IRU WKH SUHVHQFH RI YLUDO 51$ XVLQJ QXFOHLF DFLG DPSOLĺFDWLRQ WHFKQLTXHV FDQ EH GRQH RQ QXFKDO VNLQ DQG RWKHU samples obtained postmortem (7, 10, 14, 15)+RZHYHUWHVWVIRUGHWHFWLRQRIYLUDO 51$FRQGXFWHGRQVDPSOHVRWKHUWKDQEUDLQWLVVXHDUHOHVVVHQVLWLYHDQGFDQQRW UXOHRXWDGLDJQRVLVRIUDELHVLQDOOFDVHV$VXJJHVWHGDOJRULWKPIRUSRVWPRUWHP GLDJQRVLVLVLOOXVWUDWHGLQ)LJ Conclusions Laboratory diagnosis should be systematically applied to any suspected or SUREDEOHFDVHRIKXPDQUDELHV6XEVWDQWLDOWHFKQLFDOSURJUHVVKDVEHHQDFKLHYHG during the past few decades, and validated techniques using various clinical VSHFLPHQV DUH QRZ DYDLODEOH IRU KXPDQ UDELHV GLDJQRVLV +RZHYHU DFFHVV WR ODERUDWRU\ WHVWLQJ UHPDLQV FRQVWUDLQHG LQ VHYHUDO UHVRXUFHOLPLWHG DUHDV RI WKH world in which canine rabies is endemic and where most cases of human rabies FRQWLQXHWRRFFXU7KHUHLVDQHHGWKHUHIRUHWRGHYHORSDQGLPSOHPHQWQHZWRROV VXLWDEOHIRUXVHLQGHFHQWUDOL]HGODERUDWRULHVRURQVLWHLQUHPRWHUDELHVHQGHPLF DUHDVWRHQVXUHUHOLDEOHGDWDRQKXPDQUDELHV s Antemortem and postmortem tests for diagnosis of human rabies Laboratory techniques in rabies Fifth edition Part 1. General considerations 7DEOH+XPDQUDELHVSRVWPRUWHPGLDJQRVWLFWHVWV Test Target Clinical Advantages Limitations Additional Comments samples 'LUHFWĻXRUHVFHQW 9LUDODQWLJHQ %UDLQWLVVXH 5DSLGWXUQDURXQG )UHVKEUDLQ q*ROGVWDQGDUGr antibody test WLPH tK HDV\ tissue ideal for for postmortem ')$7 to perform; almost testing; results FRQĺUPDWLRQRIUDELHV VHQVLWLYH on autolysed or on brain tissue DQGVSHFLĺF decomposed tissues less 9DOLGDWHGDQG reliable commercially available UHDJHQWV FRQMXJDWHG Obtaining antibodies) postmortem brain tissue challenging Need for expensive ĻXRUHVFHQFH PLFURVFRSHDQGVNLOOHG personnel to interpret test results limits its use in many developing countries ')$7RQQXFKDOVNLQ biopsy obtained postmortem can be done when brain tissue is not available, but sensitivity is low; negative test result does not rule out rabies Direct rapid immu 9LUDODQWLJHQ %UDLQWLVVXH 5DSLGWXUQDURXQG 5HDJHQWVQRW 5HFRPPHQGHGDVDQ nohistochemistry WLPH tK commercially OIE diagnostic test for WHVW '5,7 sensitivity and available; complex postmortem diagnosis VSHFLĺFLW\ protocol (multiple of rabies in animals comparable to wash steps) ')$7 UHTXLUHV Not adequately only light validated for human microscope for rabies diagnosis interpretation Histological Inclusion %UDLQWLVVXH Easy and rapid Less sensitive No longer LGHQWLĺFDWLRQRI bodies WHFKQLTXH 6HOOHUpV than immunohis recommended for q1HJULERGLHVrE\ (aggregates of staining); use of tochemistry primary diagnosis, both 6HOOHUpVWHFKQLTXH viral particles) IRUPDOLQĺ[HG methods, espe in humans and animals on smears from tissues for H&E cially in autolysed fresh brain tissue staining reduces specimens Negative test result or by H&E staining biohazard does not rule out rabies on sections H&E staining on RIIRUPDOLQ IRUPDOLQĺ[HG ĺ[HGSDUDIĺQ WLVVXHVLVWLPH embedded tissues FRQVXPLQJ WDNHV tGD\V 5DSLG 9LUDODQWLJHQ %UDLQWLVVXH 5DSLGWXUQDURXQG Need for Not adequately LPPXQRFKURPDWR WLPH tPLQ much better validated for human graphic diagnostic low technological standardization UDELHVGLDJQRVLV WHVW 5,'7 requirement; can and quality control be used at point of of commercially +HOSIXOIRUUDSLGSRVW sampling; suitable DYDLODEOHNLWV mortem diagnosis for diagnosis in variable sensitivity of animal rabies in ĺHOGFRQGLWLRQV DQGVSHFLĺFLW\ developing countries NLWVFRPPHUFLDOO\ reported where surveillance is available ODFNLQJ Antemortem and postmortem tests for diagnosis of human rabies Laboratory techniques in rabies Fifth edition Part 1. General considerations 7DEOH&RQWLQXHG Test Target Clinical Advantages Limitations Additional Comments samples (Q]\PHOLQNHG 9LUDODQWLJHQ %UDLQWLVVXH High sensitivity 5HDJHQWVQRW Can be used as immunosorbent DQGVSHFLĺFLW\ commercially a complementary assay (ELISA) rapid; can analyse available technique several samples simultaneously Nucleic acid 9LUDO51$ %UDLQWLVVXH 5DSLGWXUQDURXQG Quality of clinical Amplicons from DPSOLĺFDWLRQ WLPH tK specimen vital; conventional (not (NAA) sensitive and appropriate UHDOWLPH 3&5FDQ VSHFLĺF storage and be used for genetic Conventional and transport characterization UHDOWLPHUHYHUVH conditions must transcriptase be maintained 5HTXLUHPHQWRI polymerase chain expensive infrastructure UHDFWLRQ3&5 1XFKDOVNLQ 5DSLGWXUQDURXQG Low sensitivity and trained manpower 573&5 DVZHOO ELRSV\KDLU WLPH tK compared with and stringent quality as other methods follicles, brain tissue; control &6) should be tested only if brain tissue 5LVNRIDPSOLFRQ is unavailable; contamination leading negative test to false positive results result cannot rule higher in conventional out rabies in all 3&5 cases 5DELHVWLVVXH Live virus %UDLQWLVVXH enables Long turnaround 9LUXVLVRODWLRQ culture infection propagation WLPH t techniques are not WHVW 57&,7 of virus and days); requires feasible for use as is valuable for ĻXRUHVFHQFH routine diagnostic tests; obtaining large microscope, they can be done only in quantities of virus cell culture specialized laboratories for molecular and adequate IRUFRQĺUPDWRU\ characterization biocontainment diagnosis, and more and other studies facilities as well as importantly for further operator expertise characterization of viruses 0RXVHLQRFXODWLRQ Live virus %UDLQWLVVXH Enables 9HU\ORQJ 7KH0,7VKRXOGEH WHVW 0,7 propagation turnaround UHSODFHGE\WKH57&,7 of virus and WLPH XSWR whenever possible is valuable for days); requires obtaining large animal facility quantities of virus and specialized for molecular laboratory characterization with adequate and other studies biocontainment; ethically challenging 5DSLGĻXRUHVFHQW 9LUXV %ORRG Diagnostic 5HVXOWVGLIĺFXOW Negative test result focus inhibition neutralizing (serum), of rabies in to interpret does not rule out rabies, WHVW 5)),7 antibodies &6) individuals not in previously since antibodies appear 5)),7)$91 vaccinated vaccinated late in the course of )OXRUHVFHQW or antibodies previously individuals LOOQHVV !GD\V antibody virus against rabies neutralization test glycoprotein )$91 (ELISA) ELISA &6)FHUHEURVSLQDOĻXLG + (KDHPDWR[\OLQDQGHRVLQ 2,(:RUOG2UJDQLVDWLRQIRU$QLPDO+HDOWK Antemortem and postmortem tests for diagnosis of human rabies Laboratory techniques in rabies Fifth edition Part 1. 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General considerations Chapter 6 Histopathological techniques in the laboratory diagnosis of human rabies Introduction Histopathological examination of the brain is one of the oldest techniques for the GLDJQRVLVRIUDELHVDQGZDVWKHJROGVWDQGDUGIRUFRQĺUPDWLRQRIUDELHVGLDJQRVLV EHIRUHWKHDGYHQWRIPRUHDGYDQFHGWHFKQLTXHVVXFKDVLPPXQRĻXRUHVFHQFH LPPXQRKLVWRFKHPLVWU\ DQG PROHFXODU PHWKRGV 6XFK HYDOXDWLRQ UHTXLUHV FORVH DWWHQWLRQWRHYHU\VWHSIURPEUDLQUHPRYDOĺ[DWLRQVDPSOLQJIRUKLVWRSDWKRORJ\ WLVVXHSURFHVVLQJVHFWLRQLQJDQGVWDLQLQJ(DFKRIWKHVHVWHSVLVGHWDLOHGEHORZ $RCKNĂWCěKONMGěJOFS 5ěGP The brain begins to disintegrate as soon as it is disconnected from its blood VXSSO\7KHSURFHVVRIDUUHVWLQJWKLVSURFHVVRIGHWHULRUDWLRQDQGSUHVHUYLQJWKH PRUSKRORJ\RIEUDLQWLVVXHLVFDOOHGqĺ[DWLRQr7KHWZRPRVWFRPPRQZD\VRI ĺ[LQJWKHEUDLQDUHIUHH]LQJDQGWKHXVHRIĺ[DWLYHVROXWLRQV(DFKPHWKRGKDVLWV DGYDQWDJHVDQGGLVDGYDQWDJHV )UHH]LQJ LV D PXFK TXLFNHU ZD\ RI ĺ[LQJ WKH EUDLQ WKDQ XVLQJ ĺ[DWLYH VROX WLRQV}(1) and tends to preserve the biochemical and molecular aspects, but it is QRWDSSURSULDWHIRUKLVWRORJ\DQGLPPXQRF\WRFKHPLVWU\7ZRFRPPRQPHWKRGV of rapidly freezing the brain include the use of dry ice and snap freezing in liquid QLWURJHQXVLQJSUHFRROHGLVRSHQWDQH)UHH]LQJRIWLVVXHVGHVWUR\VPRUSKRORJ\ DQGWKHUHE\SUHFOXGHVKLVWRSDWKRORJLFDOH[DPLQDWLRQIRUGLDJQRVLV,WLVLGHDOIRU HPSOR\LQJLPPXQRĻXRUHVFHQFHRQVPHDUVDQGPROHFXODUPHWKRGVEXWUHQGHUV WLVVXHV XQVXLWDEOH IRU KLVWRSDWKRORJ\ 7KH IRUPHU SHUPLWV H[DPLQDWLRQ RI RQO\ limited amounts of brain tissues, whereas histopathology allows sampling and GHWDLOHGH[DPLQDWLRQRIDOOQHXURDQDWRPLFDODUHDVLQWKHEUDLQ )L[HGEUDLQWLVVXHVDUHLGHDOIRUKLVWRSDWKRORJLFDOH[DPLQDWLRQDQGLPPXQRKLV WRFKHPLVWU\ 7KH PRVW FRPPRQO\ XVHG ĺ[DWLYHV LQFOXGH QHXWUDO EXIIHUHG IRUPDOLQJOXWDUDOGHK\GHRUDOFRKRO)L[LQJWKHEUDLQZLWKĺ[DWLYHVROX WLRQVWDNHVORQJHUWKDQIUHH]LQJEXWWKHEUDLQLVPXFKPRUHUHVLOLHQWHDVLHUWR KDQGOHDQGSURYLGHVH[FHOOHQWPRUSKRORJ\$IRUPDOLQVROXWLRQLVDGHTXDWH DQGLGHDOIRUSHUIRUPLQJDOOKLVWRORJLFDOVWDLQVLQFOXGLQJLPPXQRF\WRFKHPLVWU\ Histopathological techniques in the laboratory diagnosis of human rabies Laboratory techniques in rabies Fifth edition Part 1. General considerations 0DWHULDOVUHTXLUHGIRUEUDLQWLVVXHğ[DWLRQ(1) Reagents s IRUPDOGHK\GHt • distilled water • sodium phosphate, monobasic • sodium phosphate, dibasic (anhydrous) Preparation and storage of reagents 3UHSDUDWLRQRIQHXWUDOEXIIHUHGIRUPDOLQVROXWLRQ s IRUPDOGHK\GHt P/ s GLVWLOOHGZDWHU P/ s VRGLXPSKRVSKDWHPRQREDVLF J s VRGLXPSKRVSKDWHGLEDVLF DQK\GURXV J The salts are dissolved in a mixture of formaldehyde and water, with continuous VWLUULQJ7KHS+VKRXOGEHEHWZHHQDQG7KHð[DWLYHVROXWLRQVKRXOGEH stored in a container labelled “HAZARDOUS CHEMICAL”. Precautions while handling:$YRLGFRQWDFWDQGLQKDODWLRQZRUNLQDZHOOYHQWL lated area and wear protective gloves (2) Protocol Whole brain )L[ WKH EUDLQ UHPRYHG DW SRVWPRUWHP IROORZLQJ FUDQLRWRP\ LQ QHXWUDO EXIIHUHGIRUPDOLQ 6XVSHQGWKHEUDLQLQDEXFNHWFRQWDLQLQJIRUPDOLQE\SDVVLQJDWKUHDG XQGHUWKHEDVLODUDUWHU\ )LJ (QVXUHWKDWWKHEUDLQUHPDLQVVXVSHQGHGIRUDPLQLPXPSHULRGRItZHHNV DQGWKDWLWGRHVQRWFRPHLQWRFRQWDFWZLWKWKHVLGHVRUERWWRPRIWKHEXFNHW 7KLVDVVXUHVRSWLPXPĺ[DWLRQDYRLGLQJGLVWRUWLRQRIWKHEUDLQ $VOLWFDQEHPDGHLQWKHFRUSXVFDOORVXPWRHQVXUHEHWWHUIRUPDOLQSHQHWUDWLRQ Precaution:8VHDWOHDVW/RIIRUPDOLQWRĺ[WKHHQWLUHEUDLQIRUDPLQLPXP SHULRGRItZHHNV Brain biopsies )L[WKHELRSVLHVLQQHXWUDOEXIIHUHGIRUPDOLQHQVXULQJWKHYROXPHLVDW OHDVWWLPHVWKDWRIWKHWLVVXHVL]H ,PPHUVHWKHWLVVXHLQDZLGHQHFNERWWOH)RUWLVVXHVDPSOHVRItPPKRI ĺ[DWLRQLVVXIĺFLHQW/DUJHUVSHFLPHQVQHHGWREHVOLFHGLQWRWKLQQHUVHFWLRQV tFP GXULQJJURVVLQJDQGOHIWRYHUQLJKWIRUSURSHUĺ[DWLRQDVWKHSHQH WUDWLRQUDWHIRUIRUPDOLQLVORZ tPPK Histopathological techniques in the laboratory diagnosis of human rabies Laboratory techniques in rabies Fifth edition Part 1. General considerations WHRI0HQWDO+HDOWKDQG1HXURVFLHQFHV %DQJDORUH,QGLD %\FRXUWHV\RI$QLWD0DKDGHYDUQDQG5LWD0DQL1DWLRQDO,QVWLWX )LJ,GHDOPHWKRGIRUĺ[LQJWKHEUDLQE\VXVSHQVLRQLQQHXWUDOEXIIHUHGIRUPDOLQ using a thread suspended under the basilar artery to prevent tissue distortion 5CMPĚKNIěJGDRCKNHORPROEGSSKNI 5ěGP 2QFHWKHEUDLQLVĺ[HGRSWLPDOO\LWLVVOLFHGVHULDOO\LQWKHFRURQDOSODQHLQWRFP WKLFNVODEVDQGWKHEUDLQVWHPLQWKHKRUL]RQWDOSODQH7KHJURVVDOWHUDWLRQVLIDQ\ DUHUHFRUGHG7KHGLIIHUHQWQHXURDQDWRPLFDODUHDVDUHVDPSOHGDVSHUWKHVWDQ GDUGWHPSODWHDVVKRZQ )LJ Histopathological techniques in the laboratory diagnosis of human rabies Laboratory techniques in rabies Fifth edition Part 1. General considerations 0LQLPXPVHWRIEUDLQ sections for establishing the diagnosis of rabies VXSHULRUIURQWDO gyrus RUELWRIURQWDOJ\UXV FLQJXODWHJ\UXV EDVDOJDQJOLD DP\JGDOD WKDODPXV SRVWHULRU hippocampus and parahippocampal gyrus LQIHULRUSDULHWDO lobule VWULDWHFRUWH[ PLGEUDLQ SRQV FHUHEHOOXP (including the dentate nucleus) PHGXOOD FHUYLFDOFRUG %\FRXUWHV\RI$QLWD0DKDGHYDUQDQG5LWD0DQL1DWLRQDO,QVWLWXWHRI 0HQWDO+HDOWKDQG1HXURVFLHQFHV%DQJDORUH,QGLD )LJ1HXURDQDWRPLFDOVLWHVWREHVDPSOHGIURPEUDLQVOLFHV Histopathological techniques in the laboratory diagnosis of human rabies Laboratory techniques in rabies Fifth edition Part 1. 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General considerations Chapter 7 Brain removal Introduction Techniques for postmortem laboratory diagnosis of rabies in animals are FRQGXFWHGRQEUDLQPDWHULDO7KHEUDLQWLVVXHFDQEHFROOHFWHGHLWKHULQDQHFURSV\ URRPDIWHURSHQLQJWKHVNXOODQGUHPRYLQJWKHHQWLUHEUDLQRULQWKHĺHOGZLWKRXW RSHQLQJWKHVNXOOE\VDPSOLQJWKHWLVVXHWKURXJKWKHIRUDPHQPDJQXPRUWKURXJK WKHERWWRPRIWKHH\HVRFNHW%RWKWHFKQLTXHVDUHSUHVHQWHGKHUH General requirements Staff in contact with suspect specimens should be vaccinated against rabies ZLWKDOLFHQVHGYDFFLQHDQGPXVWIROORZDOOELRVDIHW\UHFRPPHQGDWLRQV,GHDOO\ GHSHQGLQJXSRQWKHUHODWLYHULVNRIYLUDOH[SRVXUHWKHVWDIIVKRXOGEHVHURORJLFDOO\ VDPSOHGHYHU\PRQWKVWR\HDUVDQGUHFHLYHDERRVWHUGRVHRIYDFFLQHLIWKH YLUXVQHXWUDOL]LQJDQWLERG\WLWUHIDOOVEHORZ,8P/RIVHUXP(1) :KLFKHYHUPHWKRGLVFKRVHQVRPHFRPPRQUHTXLUHPHQWVH[LVW s 8VHRQHVHWRILQVWUXPHQWVSHUDQLPDOWRDYRLGDQ\FURVVFRQWDPLQDWLRQRI VSHFLPHQV • Clean and decontaminate any contaminated instruments chemically (2, 3) or WKHUPDOO\ HJERLOLQJLQZDWHU s 'HFRQWDPLQDWH DOO ELRORJLFDO ZDVWH LGHDOO\ E\ LQFLQHUDWLRQ :KHUH WKLV LV QRW SRVVLEOH WKH ZDVWH VKRXOG EH EXULHG LQ TXLFNOLPH LQ DQ LVRODWHG SODFH selected to avoid any water pollution and at a depth that cannot be reached by FDUQLYRUHV6DIHGLVSRVDORIWKHVHZDVWHVLVDQLPSRUWDQWDVSHFWRIODERUDWRU\ PDQDJHPHQW:DVWHVKRXOGEHGLVSRVHGRILQFRPSOLDQFHZLWKORFDOOHJLVODWLRQ s &OHDQDQ\DSSDUDWXVXVHGLQWKHSRVWPRUWHPURRP WDEOHWRSVYLFHH\HVRFNHW IRUFHSV HWF EHWZHHQ VSHFLPHQV $OO VXUIDFHV DQG LQVWUXPHQWV VKRXOG EH ZDVKHGDQGGLVLQIHFWHGDIWHUHYHU\GLDJQRVWLFVHVVLRQDQGWKHĻRRUZDOOVDQG RWKHUVWUXFWXUHVRIWKHSRVWPRUWHPURRPGLVLQIHFWHGDQGZDVKHGGRZQGDLO\ ,Q WKH ODERUDWRU\ ZHDULQJ SURWHFWLYH FORWKLQJ HJ JRZQV DSURQV JORYHV YLVRUVPDVNVJRJJOHVDQGERRWV ZLOOSURWHFWWKHRSHUDWRUpVVNLQDQGPXFRXV PHPEUDQHV DJDLQVW VSODVKLQJ DQG VSLOODJH $OO SURWHFWLYH FORWKLQJ PXVW EH UHPRYHGZKHQOHDYLQJWKHUDELHVODERUDWRU\DQGNHSWQHDUWKHH[LW,QWKHĺHOG use of PPE, including gloves, apron or gown and goggles, is a minimum require PHQW 5$%9 LV HDVLO\ LQDFWLYDWHG E\ KHDW RU FKHPLFDO WUHDWPHQW ,RGLQH TXDWHU QDU\DPPRQLXPFRPSRXQGVDQGGLVLQIHFWDQW HJ9LUNRQ DUHVRPHRIWKHPRVW Brain removal Laboratory techniques in rabies Fifth edition Part 1. General considerations FRPPRQO\ HPSOR\HG GLVLQIHFWDQWV +RZHYHU DWWHQWLRQ PXVW EH GUDZQ WR WKH decreasing activity of some disinfectants as quaternary ammonium compounds in WKHSUHVHQFHRIKDUGZDWHURUODUJHDPRXQWVRIRUJDQLFPDWWHU,IPROHFXODUWHFK QLTXHVDUHWREHXVHGWKHVHOHFWHGGLVLQIHFWDQWPXVWQRWRQO\LQDFWLYDWH5$%9EXW also disrupt nucleic acids (2) Brain removal in the laboratory necropsy room Materials s VFDOSHOVNQLYHV • dissection forceps s KHDGĺ[DWLRQV\VWHPVXFKDV DYLFHZLWKSDUDOOHOFODPSLQJMDZV IRUFHSVWKDWDOORZVWDELOL]DWLRQRIWKHKHDGWKURXJKWKHH\HVRFNHWV 7RRSHQWKHVNXOOZKDWHYHUWKHV\VWHPXVHGLWVKRXOGEHSRVVLEOHWRIROORZWKH SULQFLSOHqRQHEODGHVDZRQHKHDGrZLWK DVWURQJEODGHZLWKDKDPPHU DPHGLFDOSODVWHUYLEUDWLQJVDZ DKDQGEXWFKHUVDZ • disposable or sterile spoons • Petri dishes 6NLQUHĻHFWHG 7HPSRUDOPXVFOHVUHPRYHG :LQGRZRSHQHGLQWKHVNXOO )LJ2SHQLQJWKHVNXOO Brain removal Laboratory techniques in rabies Fifth edition Part 1. General considerations Protocol 7ZRPDLQPHWKRGVRIEUDLQUHPRYDOLQWKHODERUDWRU\H[LVW Opening a section of the skull (Fig. 7.1) 6WDELOL]HWKHKHDGĺUPO\ &XWWKHVNLQDORQJDPHGLDQOLQHRQWKHWRSRIWKHKHDGDQGUHĻHFWLW 5HPRYHWKHWHPSRUDOPXVFOHV 2SHQDZLQGRZLQWKHVNXOOWRDFFHVVWKHEUDLQ 5HPRYHDQ\GXUDPDWHUWKDWLVVWLOODGKHUHQWWRWKHEUDLQ 8VH D VSRRQ WR FDUHIXOO\ LVRODWH WKH EUDLQ IURP WKH VNXOO DQG VFDOSHO ZLWK IRUFHSVWRFROOHFWWKHFHUHEHOOXPDQGPHGXOODREORQJDWD 7UDQVIHUWKHFHQWUDOQHUYRXVV\VWHPWRWKHGLVVHFWLRQWDEOHIRUVDPSOLQJ Cutting a longitudinal section of the head 0DLQWDLQĺUPO\WKHKHDGLQDYLFH 8VLQJDEXWFKHUpVVDZFXWWKHKHDGORQJLWXGLQDOO\ &ROOHFWWKH&16IURPHDFKKDOIKHDG 7UDQVIHUWKH&16WRWKHGLVVHFWLRQWDEOHIRUVDPSOLQJ $RCKNRGMOUCĚKNěJGĂGĚF %UDLQ VDPSOLQJ E\ RSHQLQJ WKH VNXOO LV KD]DUGRXV ZKHQ FRQGXFWHG RXWVLGH RI WKH ODERUDWRU\ +RZHYHU LQ ĺHOG FRQGLWLRQV LW PD\ EH QHFHVVDU\ WR FROOHFW samples of brain tissue for rabies diagnosis; this can be done without opening WKHVNXOOHLWKHUWKURXJKWKHIRUDPHQPDJQXPRUWKURXJKWKHSRVWHULRUZDOORIWKH H\HVRFNHW The examples given below are for animals the size of a dog, and the tools used PXVWEHDGDSWHGWRWKHVL]HRIWKHDQLPDO 'LDJQRVLV ZLWK UHIHUHQFH PHWKRGV SHUIRUPHG RQ D IXOO WKLFNQHVV F\OLQGULFDO specimen of brain tissue collected via the foramen magnum is as reliable tech QLTXHDVWKDWSHUIRUPHGRQWKHKLSSRFDPSXVDIWHURSHQLQJWKHVNXOODQGGLVVHF ting the brain (4)(LWKHUWKLVWHFKQLTXHRUDYHQWUDODSSURDFKYLDWKHWKURDWFDQEH XVHGWRDFFHVVWKHVSLQDOFRUGDQGEUDLQVWHPDVGHVFULEHGLQWKH'5,7SURWRFRO (Chapter 12 Materials s tP/SLSHWWHV s GULQNLQJVWUDZV łPPLQGLDPHWHU • scalpels s WUDQVSRUWWXEHVZLWKRUZLWKRXWDĺHOGSUHVHUYDWLYHVROXWLRQ HJJO\FHUROt saline) Brain removal Laboratory techniques in rabies Fifth edition Part 1. General considerations Foramen magnum sampling procedure %UDLQ VDPSOHV PD\ EH WDNHQ WKURXJK WKH IRUDPHQ PDJQXP (5) by using a SODVWLFGULQNLQJVWUDZ łPPLQGLDPHWHU RUDP/GLVSRVDEOHSODVWLFSLSHWWH (6)7KH}VWHSVRIWKLVSURFHGXUHDUHVXFFHVVLYHO\ 1. Access to the foramen magnum a) of the entire animal: L %HQGWKHKHDGWRH[SRVHWKHRFFLSLWDOUHJLRQ LL &XWWKHVNLQDQGQHFNPXVFOHVRYHUWKHMRLQWEHWZHHQWKHRFFLSLWDO ERQHDQGWKHDWODVYHUWHEUD LLL 2SHQWKHDWODQWRRFFLSLWDOMRLQWE\FXWWLQJWKHGRUVDOPHPEUDQH DQGWKHPHQLQJHVWRDFFHVVIRUDPHQPDJQXP E RIWKHKHDGDORQH FKHFNWKDWWKHIRUDPHQPDJQXPLVHDVLO\DFFHVVLEOH 2. Collection of brain samples (Fig. 7.2) a) Enter the straw through the foramen magnum and turn it while SXVKLQJLWWRZDUGVRQHH\H%\URWDWLQJDQGVFUHZLQJWKHVWUDZWKLV way, samples can be collected successively from the brainstem, base RIWKHFHUHEHOOXPKLSSRFDPSXVWKDODPXVDQGIURQWDOFRUWH[ Note:%HFDUHIXOQRWWRSXVKWKHVWUDZWRRFORVHWRWKHWRSRIWKHVNXOO EHFDXVHLWPD\WKHQEHEORFNHGE\WKHVHFWLRQRIGXUDPDWHUWKDW FRYHUVWKHFHUHEHOOXP E 3LQFK WKH VWUDZ EHWZHHQ WKH ĺQJHUV DQG JHQWO\ ZLWKGUDZ LW ZKLOH FRQWLQXLQJWRWXUQLW ÆYLOOH)UDQFH %HQGLQJWKHKHDGWRH[SRVHWKHDWODQWRRFFLSLWDOMRLQW $FFHVVLQJWKHMRLQW %\FRXUWHV\RI-DFTXHV%DUUDWDQG)ORUHQFH&OLTXHW$16(60DO] Straw sampling Area sampled by the straw )LJ6WUDZVDPSOLQJLQDGRJ Brain removal Laboratory techniques in rabies Fifth edition Part 1. General considerations 3. Transfer of the sample a) If the sample is to be analysed immediately, the cylinder of brain tissue is processed according to the operating instructions of the WHVW+HQFHLIWKHWHVWXVHVWKHEUDLQVWHPLWVORFDWLRQLVQRWHGLQWKH WXEH b) If the sample must be sent to a laboratory, the straw, with the sample LQVLGHLVFXWLQWKHWUDQVSRUWWXEH,IDUDSLGWUDQVSRUWZLWKFROGSDFNV LVQRWDYDLODEOHDJO\FHULQEDVHGVDOLQHSUHVHUYDWLYHVROXWLRQPD\EH XVHG LWLVWKHQLPSRUWDQWWROHWWKHEUDLQWLVVXHLQVLGHWKHVWUDZ 8CRKCěKON SCMPĚKNIěJROTIJěJGGXGSOELGě &HUHEUDOWLVVXHVVDPSOHGWKURXJKWKHH\HVRFNHWDUHFROOHFWHGLQWKHVDPHZD\ EXWWKH\DUHWDNHQLQUHYHUVHRUGHU(7) 3XVKWKHH\HEDOOWRWKHRXWHUVLGHRIWKHH\HVRFNHW ,QWURGXFH D WURFDU LQ WKH LQQHU DQJOH RI WKH RUELW WR PDNH DQ HQWU\ WKURXJKWKHSRVWHULRUZDOORIWKHH\HVRFNHW ,QWURGXFHWKURXJKWKLVKROHDSODVWLFGULQNLQJVWUDZRUDP/GLVSR sable plastic pipette, screwing it in the direction of the foramen PDJQXP 3LQFKWKHVWUDZEHWZHHQWKHĺQJHUVDQGJHQWO\ZLWKGUDZLWIURPWKH KHDG 5CĚKUCRXIĚCNFRGMOUCĚ )RU HSLGHPLRORJLFDO VWXGLHV WKH SUHVHQFH RI 5$%9 LQ VDOLYDU\ JODQGV RU LWV DQWLJHQVRUQXFOHLFDFLGDOORZVDQHYDOXDWLRQRIWKHUROHRIUHVHUYRLUDQGqQRQUH servoir” species in the dissemination of rabies and provides an additional source RIPDWHULDOIRULVRODWLRQ )LJ :LWKWKHYHQWUDOVLGHRIWKHQHFNXSPDNHDORQJLWXGLQDOVHFWLRQRIWKH VNLQRYHUWKHWUDFKHDDQGUHĻHFWLW 7KHPDQGLEXODUVDOLYDU\JODQGLVDZHOOGHĺQHGJODQGXQGHUWKHPDQGL bular lymph node, behind the posterior wall of the masseter muscle DQGXQGHUWKHMXJXODUYHLQ 7KHSDURWLGVDOLYDU\JODQGLVQRWDVZHOOGHĺQHGDVWKHPDQGLEXODU RQHO\LQJRQWKHHDUGXFW )LJ'LVSRVLWLRQRIFDQLQHPDQGLEXODUDQGSDURWLGVDOLYDU\JODQGV Brain removal Laboratory techniques in rabies Fifth edition Part 1. General considerations References :+2([SHUW&RQVXOWDWLRQRQ5DELHVWKLUGUHSRUW*HQHYD :RUOG+HDOWK2UJD QL]DWLRQ :+2 7HFKQLFDO 5HSRUW 6HULHV 1R KWWS DSSVZKRLQW LULVELWVWUHDPKDQGOH HQJSGI DFFHVVHG 0D\ $LHOOR5=HFFKLQ%7LR]]R&DHQD]]R6&DWWROL*GH%HQHGLFWLV3'LVLQIHF tion protocols for necropsy equipment in rabies laboratories: safety of personnel DQG GLDJQRVWLF RXWFRPH - 9LURO 0HWKRGV t GRL M MYLURPHW :X * 6HOGHQ ' )RRNV $5 %DQ\DUG $ ,QDFWLYDWLRQ RI UDELHV YLUXV - 9LURO 0HWKRGV tGRL MMYLURPHW %DUUDW-+DOHN+>6LPSOLĺHGDQGDGDSWHGVDPSOLQJDQGFRQVHUYDWLRQWHFK QLTXHVIRUWKHGLDJQRVLVRIUDELHVLQ0HGLWHUUDQHDQFRXQWULHV@0DJKUHE9ÆW t LQ)UHQFK %DUUDW-%ODQFRX-6LPSOLĺHGWHFKQLTXHIRUWKHFROOHFWLRQVWRUDJHDQGVKLS PHQWRIEUDLQVSHFLPHQVIRUUDELHVGLDJQRVLV*HQHYD :RUOG+HDOWK2UJDQL]DWLRQ :+25DE5HV KWWS ZZZZKRLQWUDELHVHQVLPSOLĺHGBWHFKQLTXHB FROOHFWLRQBEUDLQBVSHFLPBUDELHVBGLDJQRVLVSGIDFFHVVHG$SULO %RXUK\+6XUHDX3>/DERUDWRU\PHWKRGVIRUWKHGLDJQRVLVRIUDELHV@3DULV ,QVWLWXW3DVWHXU LQ)UHQFK 0RQWDQR+LURVH0$%RXUK\+6XUHDX35HWURRUELWDOURXWHIRUEUDLQVSHFLPHQ FROOHFWLRQRIUDELHVGLDJQRVLV9HW5HF t Brain removal Laboratory techniques in rabies Fifth edition Part 2. Detection of virus Part 2. Detection of virus Laboratory techniques in rabies Fifth edition Part 2. Detection of virus Chapter 8 Virus isolation in animals: the mouse inoculation test Introduction Isolation of pathogens in animal models, including the mouse inoculation test 0,7 IRUUDELHVGLDJQRVLVKDVEHHQXVHGLQJOREDOODERUDWRU\SUDFWLFHIRUPRUH than a century (1)7KHDGYHQWRIFHOOWLVVXHFXOWXUHVKDVIDFLOLWDWHGDVXEVWDQWLDO UHGXFWLRQRIDQLPDOXVH8VHRIDQLPDOVLVGLVFRXUDJHGZKHQRWKHUPHWKRGVDUH DYDLODEOH+RZHYHUWKHUHLVVWLOOQRDOWHUQDWLYHWRDQLPDOXVHLQPDQ\DUHDVRILQIHF tious disease research: for example, pathogenicity studies, evaluation of safety DQGHIĺFDF\RIELRORJLFDOVDQGRWKHUSKDUPDFHXWLFDOSUHSDUDWLRQVRUWKHDEVHQFH of cell culture capacity in a given diagnostic laboratory, which is still common in developing countries (2, 3)$OWKRXJKZHDQLQJDQGDGXOWPLFHGHPRQVWUDWHOHVV susceptibility to lyssaviruses than some other mammals (such as Syrian hams ters, particularly via peripheral inoculation routes), they offer operational conve nience because of their small body size, which allows housing of several animals in a cage, relatively inexpensive husbandry, and convenient handling offered by WKHLUORQJWDLOV,QRFXODWHGLQWUDFUDQLDOO\PLFH SDUWLFXODUO\WKRVHDJHGtGD\V demonstrate high susceptibility to lyssaviruses similar to that of nerve cell tissue FXOWXUHV Isolation of lyssaviruses in mice has several advantages and disadvantages IURPLVRODWLRQLQFHOOFXOWXUHVXFKDVWKHUDSLGWLVVXHFXOWXUHLQIHFWLRQWHVW 57&,7 )RUH[DPSOHPLFHDUHXVXDOO\PRUHUHVLVWDQWWKDQRWKHUVPDOOPDPPDOVWREDFWH rial contaminants and toxic substances, which may be present in the diagnostic PDWHULDOREWDLQHGIURPGHDGDQLPDOVLQWKHĺHOG6XFKVDPSOHVIUHTXHQWO\FDQQRW be inoculated into cell culture, or inoculation of cell culture requires high dilutions RIWKHVDPSOHVZKLFKUHGXFHVWKHVHQVLWLYLW\RIWKHWHVW7KHXVHRIPLFHDOORZV DFFXPXODWLRQRIKLJKYLUXVWLWUHVDWĺUVWLQRFXODWLRQDVWKHLQIHFWLRQFDXVHVGLVHDVH RQO\DIWHUUHSOLFDWLRQRIO\VVDYLUXVHVLQWKHPRXVHEUDLQ&RQYHUVHO\PRVWFRQYHQ WLRQDOFHOOFXOWXUHVXVHGWRLVRODWHO\VVDYLUXVHVPXVWEHSDVVDJHGHYHU\tGD\V WKXV QRW SURYLGLQJ VXIĺFLHQW WLPH IRU YLUXV DPSOLĺFDWLRQ XQWLO VHYHUDO SDVVDJHV VRPHWLPHV RU PRUH KDYH EHHQ PDGH (4) 6XFK PXOWLSOH SDVVDJHV LQ YLWUR may alter virus properties due to the accumulation of mutations in viral genomes, SDUWLFXODUO\LQWKHDEVHQFHRIKRVWLPPXQHUHVSRQVHWKDWRFFXUVLQ}YLYR,QIDFW the opposite and disadvantageous consequence of long virus replication in the PRXVHEUDLQEHIRUHWKHDQLPDOEHFRPHVLOOLVWKDW0,7WDNHVORQJHUWRFRPSOHWH tZHHNV WKDQ57&,7 tSDVVDJHVtGD\VHDFK WRHQVXUHQHJDWLYHUHVXOWV In pathogen discovery studies, inoculation of animals facilitates isolation of GLIIHUHQWLQIHFWLRXVDJHQWVFDXVLQJGLVHDVHVZLWKWKHLUIXUWKHULGHQWLĺFDWLRQDQG characterization (5)6XFKSDWKRJHQVFDQEHPLVVHGLQFHOOFXOWXUHSDUWLFXODUO\LI WKH\UHSOLFDWHVORZO\DQGGRQRWSURGXFHVWURQJF\WRSDWKLFHIIHFW7KH0,7GRHV not require a sterile environment and CO incubator, and it can be performed in Virus isolation in animals Laboratory techniques in rabies Fifth edition Part 2. Detection of virus UHODWLYHO\ EDVLF ĺHOG ODERUDWRULHV ZLWK D YLYDULXP 7KHUHIRUH RSHUDWLRQDO FKRLFH EHWZHHQ0,7DQG57&,7GHSHQGVRQWKHDLPVRIWKHVWXG\WKHTXDOLW\RIVDPSOHV DQGWKHFDSDFLW\RIWKHODERUDWRU\ Choice of animals and their husbandry All conventional strains of albino laboratory mice demonstrate nearly iden WLFDOVXVFHSWLELOLW\WRO\VVDYLUXVHV6XFNOLQJPLFH DJHGtGD\V DUHSUHIHUDEOH EHFDXVHRIWKHLUKLJKVXVFHSWLELOLW\$VZDVGHPRQVWUDWHGIRUVHYHUDODWWHQXDWHG UDELHVYLUXV 5$%9 VWUDLQVPLFHDJHGtGD\VZHUHVXVFHSWLEOHWRERWKLQWUDFUD QLDODQGLQWUDPXVFXODUFKDOOHQJHZKHUHDVPLFHDJHGZHHNVZHUHQRWVXVFHS WLEOHWRWKHVHYLUXVHVLQRFXODWHGE\DQ\RIWKHURXWHV1HYHUWKHOHVVZHDQOLQJPLFH DJHGtZHHNV FDQEHXVHGIRUURXWLQHLVRODWLRQRIO\VVDYLUXVHVIURPVDPSOHV FROOHFWHGLQWKHĺHOG7KHXVHRIROGHUPLFHLVQRWUHFRPPHQGHGEHFDXVHRIWKHLU GHFUHDVHGVXVFHSWLELOLW\DQGEHFDXVHWKHLUVNXOOVDUHWRRWKLFNIRUFRQYHQWLRQDO FKDOOHQJHYLDQDUURZJDXJHQHHGOHV)RUFHGXVHRIQHHGOHVRQWKLFNVNXOOVZLOO LPPHGLDWHO\LQFUHDVHWUDXPDWLFHIIHFWV0LFHRIERWKVH[HVDUHHTXDOO\VXVFHS tible, but an attention should be paid to not put young adult mice of different VH[HVLQRQHFDJHWRDYRLGXQZDQWHGEUHHGLQJ7KHXVHRIIHPDOHVLVPRUHSUDF WLFDODVPDOHVPD\ĺJKWPRUHIUHTXHQWO\ 7KHDQLPDOVPXVWEHLQJRRGJHQHUDOKHDOWK7KLVZLOOPDNHWKHPPRUHUHVLV tant to adverse effects from intracranial inoculation and will eliminate nonspe FLĺFPRUELGLW\DQGPRUWDOLW\GXULQJWKHWHVW*HQHUDOO\DWOHDVWRUPRUHGD\VRI quarantine observation is required for mice after their delivery from a breeding facility to the diagnostic laboratory for them to adapt to the new environment, decrease any stress caused by transportation and identify any potential health SUREOHPV0LFHWKDWEHFRPHLOODQGGHPRQVWUDWHXQH[SHFWHGFOLQLFDOVLJQVGXULQJ WKHH[SHULPHQW HJGLDUUKRHD VKRXOGEHUHPRYHGLPPHGLDWHO\IURPWKHH[SH riment and submitted to the vendor or to a veterinary laboratory for diagnostic purposes (if mice have been inoculated with lyssaviruses, the laboratory where VXFKDGLDJQRVLVLVSHUIRUPHGPXVWEHOLFHQVHGIRUZRUNZLWKWKHVHSDWKRJHQV DQGGLDJQRVWLFVWDIIPXVWEHYDFFLQDWHGDSSURSULDWHO\ +RXVLQJ RI PLFH GHSHQGV RQ WKH VL]H RI WKH FDJHV XVHG ,W LV XQOLNHO\ WKDW mice can transmit lyssaviruses among each other during the observation period; WKHUHIRUH SURSHUO\ PDUNHG PLFH LQRFXODWHG ZLWK GLIIHUHQW VSHFLPHQV FDQ EH FRKRXVHG+RZHYHUWRDYRLGPLVODEHOOLQJLWLVDOZD\VSUHIHUDEOHWRNHHSLQHDFK FDJHWKHPLFHLQRFXODWHGZLWKRQO\RQHVSHFLPHQ$JURXSRItZHDQOLQJPLFH LVXVXDOO\XVHGSHUVSHFLPHQ6XFNOLQJPLFHDUHKRXVHGZLWKDGDPRQHOLWWHUSHU FDJHDQGDUHLQRFXODWHGZLWKRQHVSHFLPHQ PDUNLQJRIVXFNOLQJPLFHLVGLIĺFXOW DQGXQUHOLDEOHH[FHSWWDWWRRLQJ ,IDOLWWHULVODUJHDQGVRPHRIWKHVXFNOLQJPLFH are not used for inoculation, they should be euthanized to avoid misinterpretation RIWHVWUHVXOWV Virus isolation in animals Laboratory techniques in rabies Fifth edition Part 2. Detection of virus Preparation of the inoculum 6XVSHQVLRQVRIWLVVXHVFHOOFXOWXUHVRUVDOLYDDUHFRPPRQO\XVHGIRUWKH0,7 The tissues must be rigorously homogenized using suitable means of prepara WLRQ$OOQRQGLVSRVDEOHHTXLSPHQWXVHGIRUKRPRJHQL]DWLRQPXVWEHVWHULOL]HGE\ DXWRFODYLQJ,GHDOO\DOOVWHSVRISUHSDUDWLRQRIWKHLQRFXOXPVKRXOGEHSHUIRUPHG LQDELRVDIHW\FDELQHWWRFRQWDLQGLVSHUVLRQRIGURSOHWV %UDLQWLVVXHLVVRIWDQGFDQEHKRPRJHQL]HGHDVLO\2WKHURUJDQVSDUWLFXODUO\ WKRVHZLWKDODUJHDPRXQWRIFRQQHFWLYHWLVVXHV HJVDOLYDU\JODQGV PD\UHSUH VHQWGLIĺFXOWLHV$}SHVWOHDQGPRUWDUDUHWKHqFODVVLFDOrKRPRJHQL]DWLRQWRROVDQG FDQEHHIĺFLHQWO\XVHGLIWKHDPRXQWRIWLVVXHLVUHODWLYHO\ODUJH JRUPRUH Abrasives such as sterile sand or glass shards can be added to improve homoge QL]DWLRQRIWRXJKWLVVXHV)RUVPDOODPRXQWVRIEUDLQWLVVXHVWHULOHZRRGHQRU SODVWLFVWLFNVFDQEHXVHGWRKRPRJHQL]HWKHVDPSOHLQDPLFURWXEHZLWKDĻDWRU FRQHERWWRP tP/(SSHQGRUIRUVLPLODUWXEH DVFUHZFDSLVSUHIHUDEOHWR FRQWUROOHDNDJHV )RUVPDOODPRXQWVRIWRXJKWLVVXHVLWPD\EHQHFHVVDU\WRXVH metal or polymer beads, placed in a microtube together with the tissue sample, and VXEMHFWWKH}WXEHWRKLJKVSHHGYLEUDWLRQLQDQHOHFWULFKRPRJHQL]HUIRUt}PLQ +RPRJHQL]DWLRQVXSSOLHVLQVWUXPHQWVDQGGLOXHQWVVKRXOGEHSUHFRROHGDW& WRUHGXFHHQ]\PDWLFGHVWUXFWLRQRIYLULRQVGXULQJSUHSDUDWLRQRIWKHLQRFXOXP One of the most commonly used diluents for preparation of the suspension is a sterile isotonic NaCl (sodium chloride) solution, although mice are resistant HYHQIRULQRFXODWLRQZLWKGLVWLOOHGZDWHU$QDGGLWLRQRItRIDQLPDOVHUXPZLOO LQFUHDVHYLUXVVWDELOLW\GXULQJWKHSUHSDUDWLRQDQGLQRFXODWLRQSHULRG+RZHYHUWKH GRQRUDQLPDOIRUWKHVHUXPVXSSOHPHQWVKRXOGQHYHUEHYDFFLQDWHGDJDLQVWUDELHV If such information is unavailable, dog, cat and cattle sera should not be used for SUHSDUDWLRQRIWKHLQRFXOXP&RPPHUFLDOHTXLQHVKHHSRUUDEELWVHUDDUHXVXDOO\ VDIHIRUXVH7KHVHUXPPXVWEHKHDWLQDFWLYDWHGDW&IRUtPLQ,IVWHULOLW\ RIWKHGLOXHQWLVQRWZDUUDQWHGE\WKHPDQXIDFWXUHULWPXVWEHĺOWHUHGWKURXJKD EDFWHULDUHWDLQLQJĺOWHU0DQ\ODERUDWRULHVURXWLQHO\XVHFHOOFXOWXUHPHGLXP IRU H[DPSOHWKHPLQLPXPHVVHQWLDOPHGLXP IRUSUHSDUDWLRQRIWKHDQLPDOLQRFXOXP ,WXVXDOO\FRQWDLQVtIHWDOERYLQHVHUXPFHUWLĺHGIRUWKHDEVHQFHRI5$%9DQWL ERGLHVSDWKRJHQVDQGWR[LFVXEVWDQFHV$OWKRXJKDQWLPLFURELDODQGDQWLIXQJDO medicines are not needed to inoculate mice with fresh sterile tissue material, their use is advised for inoculation with salivary gland suspensions or other samples WKDWFDQEHFRQWDPLQDWHGZLWKEDFWHULDRUIXQJL$}PL[WXUHRISHQLFLOOLQ ,8 P/ DQGVWUHSWRP\FLQ ,8P/ KDVEHHQXVHGIRUVXFKSXUSRVHVKLVWRULFDOO\ DQGPRUHDQWLELRWLFVDUHQRZDYDLODEOHFRPPHUFLDOO\8VXDOO\DQWLELRWLFVPD\EH DOUHDG\SUHVHQWLQWKHPLQLPXPHVVHQWLDOPHGLXPDQGRWKHUFHOOFXOWXUHPHGLD $VXVSHQVLRQ E\ZHLJKW RILVW\SLFDOO\XVHGWRLQRFXODWHDQLPDOVDOWKRXJK WKLVLVDQRSHUDWLRQDOFKRLFHDQGWKHFRQFHQWUDWLRQFDQEHLQFUHDVHGIROGRU GHFUHDVHG ZLWKRXW VLJQLĺFDQW FKDQJHV LQ WKH VHQVLWLYLW\ RI WKH WHVW RU DGYHUVH HIIHFWVIRULQRFXODWHGDQLPDOV7KHUHTXLUHGDPRXQWRIGLOXHQWLVXVXDOO\DGGHGWR WKHWLVVXHVSHFLPHQJUDGXDOO\GXULQJKRPRJHQL]DWLRQ:KHQKRPRJHQL]DWLRQLV performed with beads in a closed tube, the full amount of diluent can be added WR WKH WXEH DW WKH EHJLQQLQJ $IWHU KRPRJHQL]DWLRQ WKH VXVSHQVLRQ VKRXOG EH FHQWULIXJHG WR UHPRYH JURVV SDUWLFOHV :KLOH VXFK SDUWLFOHV PD\ QRW} DGYHUVHO\ DIIHFWLQRFXODWHGDQLPDOVWKH\PD\FORJWKHV\ULQJHQHHGOHVGXULQJLQRFXODWLRQ Virus isolation in animals Laboratory techniques in rabies Fifth edition Part 2. Detection of virus &HQWULIXJDWLRQ LV XVXDOO\ SHUIRUPHG DW t g IRU t PLQ LGHDOO\ LQ D FRROLQJFHQWULIXJH & ,IEDFWHULDOFRQWDPLQDWLRQLVH[SHFWHGLQWKHVSHFLPHQ WKHVXVSHQVLRQVXSHUQDWDQWFDQDOVREHVXEMHFWHGWRĺOWUDWLRQYLDDPĺOWHU DWWDFKHGWRDV\ULQJH7KHXVHRIPĺOWHUVLVOHVVSUDFWLFDO LWGRHVQRWDGG PXFKEHQHĺWIRUUHPRYDORIEDFWHULDOFRQWDPLQDWLRQEXWDEVRUEVPLFURSDUWLFOHV of tissue that may contain the pathogen of interest in a greater quantity than the FOHDQ VXSHUQDWDQW 7KH SUHSDUHG LQRFXOXP VKRXOG EH NHSW LQ D FRQWDLQHU ZLWK SUHFRROHGLFHSDFNVRURQZHWLFHXQWLOXVH)UHH]LQJVKRXOGEHDYRLGHGEHFDXVH HDFKIUHH]HtWKDZF\FOHPD\UHGXFHYLUXVWLWUH Inoculation procedure Preparation The inoculation can be performed in a biosafety cabinet or on the laboratory EHQFK GHSHQGLQJ RQ LQVWLWXWLRQDO UHTXLUHPHQWV $V O\VVDYLUXVHV DUH QRW WUDQV PLWWHG E\ WKH DHURVRO URXWH H[FHSW XQGHU KLJKO\ VSHFLĺF FLUFXPVWDQFHV HJ D KLJKFRQFHQWUDWLRQRIDHURVROSDUWLFOHVRUDQDUWLĺFLDOO\FRQFHQWUDWHGYLUXVSUHSD UDWLRQ DQLPDOLQRFXODWLRQRQWKHEHQFKLVXVXDOO\FRQVLGHUHGVDIH7KHEHQFKDOO surfaces and instruments around must be washable; appropriate personal protec WLYHHTXLSPHQW 33( VKRXOGEHXVHGWRDYRLGVHOIH[SRVXUHDQGWRSURWHFWWKH animal from human infections that may be introduced by the operator performing WKHWHVW7KHPLQLPXP33(LQFOXGHVDODERUDWRU\FRDWZLWKORQJVHFXUHGVOHHYHV double latex gloves, a face shield and a hair net to protect against droplets of LQRFXOXP7KHUHLVDEVROXWHO\QRHIĺFLHQWZD\WRSURWHFWDQRSHUDWRUIURPVHOI VWLFN LQMXULHV E\ V\ULQJH QHHGOHV H[FHSW E\ H[HUFLVLQJ FDXWLRXV EHKDYLRXU DQG JRRGLQRFXODWLRQVNLOOV+DQGVVKRXOGQRWEHFURVVHGGXULQJWKHSURFHGXUH PLFH and mouse containers should be placed at one side, whereas syringes and sharps FRQWDLQHUVVKRXOGEHSODFHGRQWKHRWKHUVLGHIURPWKHRSHUDWRU6\ULQJHVVKRXOG QRWEHUHFDSSHGWRUHGXFHWKHULVNRIQHHGOHVWLFNLQMXULHV,IWKLVLVQRWSRVVLEOH VWDIIPXVWEHWUDLQHGLQWKHVDIHRQHKDQGFDSSLQJWHFKQLTXHRUVSHFLDOFDSSLQJ GHYLFHV DYDLODEOHLQWKHPDUNHW VKRXOGEHXVHG Anaesthesia ,GHDOO\PLFHVKRXOGEHDQDHVWKHWL]HGSULRUWRLQWUDFUDQLDOLQMHFWLRQ7KLVLVWKH RQO\RSWLRQDSSURYHGE\WKH$PHULFDQ9HWHULQDU\0HGLFDO$VVRFLDWLRQ $90$ DQG WKH$PHULFDQ$VVRFLDWLRQIRU/DERUDWRU\$QLPDO6FLHQFH $$/$6 6LPLODUUHTXLUH ments have been introduced in many countries worldwide, and their implementa WLRQLVHQIRUFHGE\WKHORFDO,QVWLWXWLRQDO$QLPDO&DUHDQG8VH3URJUDP ,$&8& ,Q JHQHUDO WKHUH DUH WZR PDMRU RSWLRQV IRU DQDHVWKHVLD RI PLFH ZLWK LQKD ODWLRQDO RU LQMHFWDEOH PHGLFLQHV 7KH PRVW FRQYHQLHQW LQKDODWLRQDO DQDHVWKHWLF XVHGIRUWKLVSXUSRVHLVLVRĻXUDQH(6),VRĻXUDQHKDVPDQ\DGYDQWDJHVRYHULQMHF table agents: minimal animal handling, large margin of safety, ease of anesthetic FRQWUROORZFRVWRIDQDHVWKHWLFDJHQWQRFRQWUROOHGPHGLFLQHVDQGTXLFNUHFR YHU\WLPHV7KHSULPDU\GLVDGYDQWDJHVDUHWKHLQLWLDOFRVWRIHTXLSPHQWDQGWKH QHHGWRFRQWUROKXPDQH[SRVXUHWRJDV ZKLFKLVDYRLGDEOHLIWKHURRPVDWLVĺHV WKHYHQWLODWLRQUHTXLUHPHQWVVWDQGDUGIRU%6/DQG%6/IDFLOLWLHV Virus isolation in animals Laboratory techniques in rabies Fifth edition Part 2. Detection of virus 0LQLPXPFRPSRQHQWVRIHTXLSPHQWLQFOXGH sLVRĻXUDQHYDSRUL]HU • supply gas (oxygen) • supply gas regulator sĻRZPHWHU tP/PLQ • induction chamber • connection tubing and valves 7KH ĻRZPHWHU VKRXOG EH VHW EHWZHHQ P/PLQ DQG P/PLQ DQG WKH} YDSRUL]HU WR 7KH LQFXEDWLRQ FKDPEHU FDQ EH SUHĺOOHG ZLWK WKH JDV DQGJURXSRIDQLPDOVSODFHGLQWKHFKDPEHU$QDHVWKHVLDW\SLFDOO\RFFXUVDIWHU t}PLQRILQFXEDWLRQDIWHUZKLFKWLPHWKHDQLPDOV LQGLYLGXDOO\ FDQEHUHPRYHG WKHRSHUDWRUWKHQKDVtPLQWRLQRFXODWHWKHPRXVHEHIRUHLWDZDNHQVZKLFK LVXVXDOO\VXIĺFLHQWWLPH8VLQJWKLVWHFKQLTXHDJURXSRIĺYHPLFHFDQEHLQRFX ODWHGGXULQJtPLQEHIRUHDQRWKHUJURXSRIPLFHLVSODFHGLQWKHFKDPEHUIRU WKHQH[WVDPSOH 2WKHULQKDODWLRQDODJHQWV KDORWKDQHVHYRĻXUDQHHWF DUHXVHGOHVVIUHTXHQWO\ Ether, used for animal and human anaesthesia historically, is not approved by $90$DQG$$/$6GXHWRLWVSRWHQWLDOVLGHHIIHFWVDQGSRRUVDIHW\FKDUDFWHULVWLFV $PRQJWKHLQMHFWDEOHDQDHVWKHWLFVNHWDPLQHLQFRPELQDWLRQZLWKRWKHUDJHQWV HJ[\OD]LQH LVXVHGPRVWIUHTXHQWO\7DEOHVKRZVH[DPSOHVRIVXFKFRPEL QDWLRQV ZKLFK DUH DGPLQLVWHUHG LQ D YROXPH RI t P/ XVXDOO\ LQWUDSHUL toneally (7)7KH}DQDHVWKHVLDXVXDOO\WDNHVHIIHFWDIWHUtPLQDQGODVWVt PLQ ,I QHHGHG WKH} DQDHVWKHWLF FDQ EH UHYHUVHG E\ DWLSDPH]ROH PJNJ EXW this is usually not necessary, because if several samples are inoculated in several JURXSVRIPLFHWKHĺUVWJURXSVZLOOZDNHXSEHIRUHWKHODVWRQHLVFRPSOHWHG Combinations of anaesthetics can be prepared in advance in designated sterile containers, which must be properly labelled and stored until the expiration date WKHHDUOLHVWDPRQJWKHPHGLFLQHVXVHGLQWKHFRPELQDWLRQ 2WKHULQMHFWDEOHDQDHVWKHWLFV EDUELWXUDWHVEHQ]RGLD]HSLQHVRSLRLGV DUHXVHG LQPLFHOHVVIUHTXHQWO\7ULEURPRHWKDQROPD\FDXVHXQSUHGLFWHGHIIHFWVLQPLFH DJHGROGHUWKDQGD\V If anaesthetics or equipment for anaesthesia are not available in the diagnostic laboratory, which frequently occurs in developing countries, or if national and insti tutional regulations do not require anaesthesia, mice may be inoculated without VHGDWLRQ DQGWKLVPHWKRGZDVXVHGIRUGHFDGHVZRUOGZLGH 2QHPXVWFRQVLGHU WKDW QHHGOHVWLFN LQMXULHV FDXVHG GXULQJ LQRFXODWLRQ DUH PRPHQWDU\ ZKHUHDV ORQJHUODVWLQJDGYHUVHHIIHFWVLIWKH\RFFXUDUHFDXVHGQRWE\WKHQHHGOHVWLFN LWVHOIEXWE\WUDXPD7KHUHIRUHDVKRUWWHUPDQDHVWKHVLDZLOOQRWSURWHFWPLFHIURP VXFKHIIHFWV,QVWHDGSUHFDXWLRQVPXVWEHWDNHQWRDYRLGH[FHVVLYHWUDXPDDQG WKHWHFKQLTXHGHVFULEHGEHORZPD\KHOSLQWKLVUHJDUG Virus isolation in animals Laboratory techniques in rabies Fifth edition Part 2. Detection of virus Medicines Dosage Comments .HWDPLQH[\OD]LQH PJNJ ǭDJRQLVWUHYHUVLEOHZLWKDWLSDPH ]ROHPJNJ .HWDPLQHPHGHWRPLGLQH )HPDOHPJNJ ǭDJRQLVWUHYHUVLEOHZLWKDWLSDPH 0DOHPJNJ ]ROHPJNJ PD\FDXVHPRGHUDWH respiratory depression 6.HWDPLQHPHGHWRPLGLQH )HPDOHPJNJPJNJ ǭDJRQLVWUHYHUVLEOHZLWKDWLSD UDFHPLFNHWDPLQHPHGHWRPLGLQH 0DOHPJNJPJNJ PH]ROHPJNJ DFWLRQVKRUWHU DQGDZDNHQLQJIDVWHUWKDQUDFHPLF NHWDPLQH .HWDPLQHGH[PHGHWRPLGLQH ttPJNJ ǭDJRQLVWUHYHUVLEOHZLWKDWLSDPH ]ROHPJNJ PD\FDXVHPRGHUDWH respiratory depression 7DEOH8VHRINHWDPLQHLQFRPELQDWLRQZLWKRWKHUPHGLFLQHVIRULQMHFWDEOHDQDHVWKHVLDRIPLFHDJHGtZHHNV Inoculation technique The volume of inoculum used for intracranial inoculation of weanling mice LV}}/ VPDOOHUYROXPHV t/ VKRXOGEHXVHGIRUVXFNOLQJPLFH,QWUDPXV FXODURUVXEFXWDQHRXVLQRFXODWLRQRIPLFHLVXVXDOO\SHUIRUPHGLQ/YROXPHV 7KHVHGRVHVFDQEHPHDVXUHGDFFXUDWHO\LQDP/LQVXOLQV\ULQJH7KHQHHGOHV VKRXOGEHtPPLQGLDPHWHU RUJDXJH DQGtFPORQJ/DUJHU needles traumatize the brain substantially, whereas larger syringes do not allow DFFXUDWHPHDVXUHPHQWRILQRFXODWLRQGRVHV,GHDOO\WKHV\ULQJHVKRXOGEHGLVSR VDEOHZLWKDQDWWDFKHGQHHGOHZKLFKGRHVQRWKDYHGHDGVSDFH7KHXVHRIVXFK D V\ULQJH ZLOO KHOS WR PLQLPL]H DLU EXEEOHV ,I EXEEOHV GR RFFXU WKH\ PXVW EH removed into a sterile soft substance (such as sterile cotton) to avoid aerosoliza WLRQ6WHULOL]HGJODVVV\ULQJHVZLWKGHWDFKDEOHQHHGOHVFDQDOVREHXVHGDOWKRXJK WKH\DUHOHVVFRQYHQLHQW,GHDOO\DQLQGLYLGXDOV\ULQJHVKRXOGEHXVHGWRLQRFXODWH HDFKPRXVH7KLVLVHVSHFLDOO\LPSRUWDQWIRUGLVSRVDEOHV\ULQJHVDVWKHLUQHHGOHV DUHPDGHRIVRIWPHWDODQGEHFRPHEOXQWDIWHUHDFKLQWUDFUDQLDOLQMHFWLRQ(YHQ}LI one syringe is used to inoculate a group of several mice, under no conditions should the same syringe be used to inoculate other samples without sterilization, WRDYRLGFURVVFRQWDPLQDWLRQ *HQHUDOO\ ULJKWKDQGHG LQGLYLGXDOV KDQGOH D PRXVH LQ WKH OHIW KDQG DQG D V\ULQJH LQ WKH ULJKW KDQG )RU VHGDWHG PLFH QR VSHFLDO KDQGOLQJ WHFKQLTXH LV QHHGHGH[FHSWWKDWWKHDQLPDOPXVWEHĺ[HGE\JUDVSLQJWKHKDLUVFUXIIDQGSUHV VLQJWKHKHDGJHQWO\EXWĺUPO\WRWKHVXUIDFHRIDQLQRFXODWLRQERDUGZKLFKZLOO DOORZDSUHFLVHQHHGOHVWLFNWRSHQHWUDWHWKHVNXOOZLWKRXWPRYLQJRUURWDWLQJWKH VNXOOXQGHUWKHSUHVVXUHRIWKHQHHGOH6HGDWHGVXFNOLQJPLFHZKLFKKDYHYHU\ VRIWVNXOOVFDQEHHDVLO\KDQGOHGE\IRUFHSVGXULQJDOOLQRFXODWLRQSURFHGXUHV 7KLVHOLPLQDWHVWKHULVNRIQHHGOHVWLFNLQMXU\IRUDQRSHUDWRU,IQRWVHGDWHGLWLV HDV\WRWDNHDPRXVHDWWKHWLSRIWKHWDLOE\WKHWKXPEDQGWKHVHFRQGĺQJHUSXW WKHDQLPDORQWKHEHQFKFOXWFKWKHEDVHRIWKHWDLOEHWZHHQWKHIRXUWKDQGĺIWK ĺQJHUVUHOHDVHWKHWKXPEDQGVHFRQGĺQJHUDQGJUDVSE\WKHPRXVHpVVFUXII 7KHPRXVHKDQGOHGWLJKWO\DWWKHEDVHRIWKHWDLODQGDWWKHVFUXIILVVXIĺFLHQWO\ Virus isolation in animals Laboratory techniques in rabies Fifth edition Part 2. Detection of virus LPPRELOL]HGWRDOORZPDQLSXODWLRQVRQWKHKHDGHYHQZLWKRXWVHGDWLRQ1RQVH GDWHGVXFNOLQJPLFHDUHW\SLFDOO\KDQGOHGRQWKHLUQHFNVFUXIIE\ĺQJHUDQGWKXPE ZKLFKZDUUDQWVVXIĺFLHQWLPPRELOL]DWLRQ 7KHV\ULQJHLVWDNHQE\WKHULJKWKDQG WKHLQGH[ĺQJHURQWKHSOXQJHUDQGWKH EDUUHOVXSSRUWHGEHWZHHQWKHWKXPEDQGWKHWKLUGDQGIRXUWKĺQJHUV DQGSXVKHG WKURXJKWKHVNXOOZLWKDTXLFNWKUXVW$OWKRXJKLQRFXODWLRQFDQEHGRQHDWGLIIH UHQWDUHDVRIWKHFDOYDULXPLWLVXVXDOO\HDV\WRPDNHWKHLQMHFWLRQVOLJKWO\DVLGH of the central line (to avoid puncture of the venous sinus), in the middle of the LPDJLQDU\OLQHEHWZHHQWKHPRXVHpVULJKWH\HDQGULJKWHDU )LJ 7KHQHHGOH VKRXOG HDVLO\ SHQHWUDWH WKH VNXOO LW VKRXOG EH LQVHUWHG t PP LQWR WKH EUDLQ WLVVXH7RRGHHSDSHQHWUDWLRQDQGLQMHFWLRQRILQRFXOXPLQWRWKHEDVDODUHDRIWKH EUDLQFDQEHYHU\WUDXPDWLF$IWHULQMHFWLQJWKHGHVLUHGYROXPHRILQRFXOXPWKH needle should be removed gently and slowly (during several seconds) to prevent UHĻX[RIWKHLQRFXOXP,IVXFKUHĻX[RFFXUVLWZLOOEHVHHQRQWKHPRXVHpVIXUDVD drop of transparent bloody liquid; it will mean that the amount of the inoculum in WKHPRXVHEUDLQLVUHGXFHG )LJ3ODFHRILQWUDFUDQLDOLQRFXODWLRQRQWKHKHDGRIDPRXVHDJHGtZHHNV $GRUVDOYLHZ %IDFLDOYLHZ Peripheral inoculation is rarely performed for diagnostic purposes as the VXVFHSWLELOLW\ RI PLFH WR O\VVDYLUXVHV LQMHFWHG YLD SHULSKHUDO URXWHV LV OLPLWHG 7KH}H[DPSOHZKHQSHULSKHUDOLQRFXODWLRQPD\EHXVHGIRUGLDJQRVWLFSXUSRVHV LVDVLJQLĺFDQWEDFWHULDOFRQWDPLQDWLRQRUWR[LFLW\RIWKHLQRFXOXPWKDWNLOOVPLFH LI LQMHFWHG LQWUDFUDQLDOO\ ,QWUDPXVFXODU FKDOOHQJH LV XVXDOO\ SHUIRUPHG LQWR WKH GHOWRLGRUJDVWURFQHPLXVPXVFOHV$QDVVLVWDQWLVQHHGHGWRVWUHWFKWKHOHJRIWKH PRXVHIRULPPRELOL]DWLRQ6XEFXWDQHRXVLQRFXODWLRQFDQEHSHUIRUPHGXQGHUWKH VFUXIIRIWKHQHFNZKHQKDQGOHGDVGHVFULEHGDERYHIRULQWUDFUDQLDOFKDOOHQJH Virus isolation in animals Laboratory techniques in rabies Fifth edition Part 2. Detection of virus &RQWDPLQDWLRQRIZRUNVXUIDFHVSURWHFWLYHZHDUDQGLQVWUXPHQWVGXULQJWKH procedure may occur via droplets of inoculum from the syringes or from the DQLPDOpVIXU7RDYRLGSRWHQWLDOH[SRVXUHDQGIXUWKHUVSUHDGRIWKHYLUXVDOOPDWH rials should be rigorously decontaminated with disinfectants immediately after FRPSOHWLRQ RI WKH LQRFXODWLRQ 2XWHU JORYHV ZKLFK DOVR FDQ EH FRQWDPLQDWHG VKRXOGEHUHSODFHGEHIRUHWKHGHFRQWDPLQDWLRQSURFHGXUH'LVSRVDEOHV\ULQJHV are discarded in sharps containers and autoclaved together with other disposable VXSSOLHV XVHG IRU LQRFXODWLRQ 1RQGLVSRVDEOH V\ULQJHV DQG QHHGOHV VKRXOG EH GLVLQIHFWHGFKHPLFDOO\ULQVHGLQZDWHUDQGDXWRFODYHGEHIRUHQH[WXVH Cages must be labelled promptly and carefully, indicating the age of the animals, the inoculation date, the inoculum used, the number of animals and any RWKHUXVHIXOLQIRUPDWLRQ,QFRPSOHWHODEHOVPD\FDXVHPLVVLQJVSHFLPHQVRUGDWD Observation and euthanasia 7KH LQRFXODWHG PLFH VKRXOG EH REVHUYHG FORVHO\ IRU a K DIWHU WKH SURFH GXUH7KLVSHULRGRIWLPHLVXVXDOO\VXIĺFLHQWIRUUHFRYHU\IURPDQDHVWKHVLDRUIURP LQRFXODWLRQWUDXPD,IPLFHFRQWLQXHWREHKDYHDEQRUPDOO\ HJVORZPRYHPHQWV DWD[LD FRQWLQXRXV RQHVLGH URWDWLRQ WKH REVHUYDWLRQ VKRXOG EH FRQWLQXHG IRU DQRWKHUKRXUDQGDGHFLVLRQRQHXWKDQDVLDLVPDGH 7KH LQFXEDWLRQ SHULRG RI 5$%9 LQ PLFH LQRFXODWHG LQWUDFUDQLDOO\ LV XVXDOO\ t}GD\VGXULQJZKLFKWLPHWKHDQLPDOVVKRXOGEHFKHFNHGWZLFHDGD\WRUHJLVWHU HDUO\FOLQLFDOVLJQV'XULQJRWKHUWLPHV VWDUWLQJIURPWKHĺUVWSRVWLQRFXODWLRQGD\ WKH\VKRXOGEHFKHFNHGDWOHDVWGDLO\7KHREVHUYDWLRQSHULRGVKRXOGH[WHQGIRU DWOHDVWGD\VXQOHVVĺ[HGVWUDLQVRI5$%9ZLWKVKRUWLQFXEDWLRQSHULRGVVXFK DVFKDOOHQJHYLUXVVWDQGDUGVWUDLQ &96 DUHXVHG7KHQXPEHURIKHDOWK\VLFN DQGGHDGDQLPDOVVKRXOGEHUHFRUGHGGDLO\RQGHVLJQDWHGFDUGVRUUHFRUGVKHHWV &OLQLFDOVLJQVVKRXOGEHUHFRUGHGIRUVLFNDQLPDOV7KHIROORZLQJFOLQLFDOVLJQVDUH suggestive of rabies: ruffed fur; tremours when handled by forceps; increased DJLWDWLRQZLWKFKDRWLFIDVWPRYHPHQWVRUMXPSV ODFNRIFRRUGLQDWLRQLQOHJVEHVW noted when a mouse is placed on a bench and starts to move; ataxia; paresis; SDUDO\VLV SURVWUDWLRQ &OLQLFDOVLJQVDQGGHDWKRFFXUULQJGXULQJWKHĺUVWGD\VDIWHULQRFXODWLRQDUH usually attributed to trauma or contamination of the inoculum with bacterial or RWKHUYLUDOSDWKRJHQV:KHQHYHUFOLQLFDOVLJQVRFFXUDGHFLVLRQRQHXWKDQDVLD VKRXOG EH PDGH SURPSWO\ EDVHG RQ WKH SDLQ VFRUHV DSSURYHG E\ WKH ,$&8& 8VXDOO\ PLOG FOLQLFDO VLJQV SDLQ VFRUHV RI t UHTXLUH LQFUHDVHG REVHUYDWLRQ ZKHUHDVVHYHUHVLJQV SDLQVFRUHVRIt UHTXLUHLPPHGLDWHHXWKDQDVLD The euthanasia is performed via intoxication with carbon dioxide or other PHDQV}(8)3ODFLQJWKHDQLPDOVLQDFKDPEHUSUHĺOOHGZLWKFDUERQGLR[LGHFDXVHV VHYHUHGLVWUHVV7KHUHIRUHWKHDQLPDOVPXVWEHSODFHGLQDFKDPEHUĺOOHGZLWK ambient air into which carbon dioxide is supplied with gradually increasing concen WUDWLRQDWDUDWHRItRIYROXPHSHUPLQ0LFHPXVWEHREVHUYHGFORVHO\WR HQVXUHHXWKDQDVLDYLDWKHDEVHQFHRIEUHDWKLQJDQGKHDUWEHDWVDQGF\DQRWLFVNLQ As alternatives to carbon dioxide, an overdose of anaesthetics used during inocu ODWLRQRUEDUELWXUDWHVFDQEHXVHG,QHYHU\FDVHHXWKDQDVLDRQDQXQFRQVFLRXV DQLPDOVKRXOGEHFRQĺUPHGE\DVHFRQGDU\PHWKRGVXFKDVFHUYLFDOGLVORFDWLRQ RUWKRUDFLFRSHQLQJ$IWHUFRPSOHWLRQRIHDFKH[SHULPHQWFDJHVPXVWEHFDUHIXOO\ FOHDQHGZLWKGLVLQIHFWDQWVDQGDXWRFODYHG Virus isolation in animals Laboratory techniques in rabies Fifth edition Part 2. Detection of virus 3RVWPRUWHPH[DPLQDWLRQ Even if mice develop neurological signs or die within the expected timeframe after inoculation, diagnosis of rabies should not be assumed without laboratory FRQĺUPDWLRQ6LPLODUFOLQLFDOVLJQVPD\EHFDXVHGE\DQXPEHURIRWKHUYLUDORU EDFWHULDOSDWKRJHQV0RVWFRPPRQO\WKHFRQĺUPDWLRQLVPDGHE\GHWHFWLRQRI O\VVDYLUXVDQWLJHQVLQWKHPRXVHEUDLQYLDWKH')$7DOWKRXJKRWKHUPHWKRGVIRU GHWHFWLRQRIDQWLJHQVRU51$FDQEHXVHGDVZHOO 5HPRYDORIWKHEUDLQVKRXOGEHSHUIRUPHGLQDELRVDIHW\FDELQHW7KHPRXVH carcass is placed on clean absorbing material (such as a gauze pad or a paper WRZHO YHQWUDOVXUIDFHGRZQZDUGV)RUDULJKWKDQGHGRSHUDWRUWKHPRXVHKHDG VKRXOGEHGLUHFWHGWRWKHOHIW7KHVNLQRIWKHQHFNDQGKHDGLVSXOOHGE\IRUFHSV DQGFXWDZD\7KHVNXOOLVJUDVSHGE\IRUFHSVLQWKHRUELWVDQGWKHFDOYDULXPLV FXWDZD\E\FXUYHGVFLVVRUVRUDVFDOSHOH[SRVLQJWKHEUDLQ7KHEUDLQLVOLIWHGE\ WKHVDPHVFLVVRUVRUVFDOSHOUHPRYHGIURPWKHVNXOODQGSODFHGRQDFOHDQJDX]H SDGRUSDSHUWRZHO$VHFWLRQRIWKHEUDLQMXVWDQWHULRUWRWKHFHUHEHOOXPLVXVHG WRPDNHLPSUHVVLRQVRQDPLFURVFRSHVOLGH Troubleshooting 8VXDOO\WKH0,7SURGXFHVDFFHSWDEOHUHVXOWVEXWLQ VRPHLQVWDQFHVLW IDLOV The most common reasons for such failures are addressed below and appropriate FRUUHFWLYHDFWLRQVDUHVXJJHVWHG If mice do not recover completely after inoculation and die shortly thereafter, this PD\UHVXOWIURPWUDXPDRUIURPWR[LFLW\RIWKHLQRFXOXP7UDXPDXVXDOO\SURGXFHV distinct morbidity or mortality patterns in different mice, whereas toxicity of the LQRFXOXPSURGXFHVDXQLIRUPUHVXOWLQDOOLQRFXODWHGDQLPDOV7RUHGXFHWUDXPDWKH needle should not be inserted too deeply during inoculation, as described above, DQG ORQJ LQDSSURSULDWHO\ JDXJHG QHHGOHV VKRXOG QRW EH XVHG $OVR \RXQJHU PLFHXVXDOO\UHFRYHUEHWWHUDIWHULQRFXODWLRQWKDQROGHUPLFH,IWKHWR[LFLW\RIWKH inoculum is the reason for failure, it should be determined whether the diluent or WKHWLVVXHVSHFLPHQLVWR[LF,IWKHGLOXHQWLVWR[LFDOOVDPSOHVSUHSDUHGZLWKLW ZLOOSURGXFHXQLIRUPPRUWDOLW\SDWWHUQV$PRFNPRXVHLQRFXODWLRQZLWKWKHVDPH GLOXHQWFDQEHSHUIRUPHGWRFRQĺUPVXFKWR[LFLW\,IWKLVLVWKHFDVHWKHGLOXHQW PXVWEHUHSODFHG,IWKHWR[LFLW\RULJLQDWHVIURPDVSHFLĺFWLVVXHVDPSOH DQGRQO\ mice inoculated with this sample demonstrate early morbidity or mortality), the VDPSOHFDQEHGLOXWHGDQGVHYHUDOIROGGLOXWLRQVFDQEHXVHGIRULQRFXODWLRQ (YHQ}WKRXJKVXFKGLOXWLRQVZLOOUHGXFHWKHVHQVLWLYLW\RIWKH0,7WKLVPD\EHWKH RQO\ZD\WRLVRODWHDSDUWLFXODUYLUXV Sometimes, mice recover after inoculation but develop clinical signs and VXFFXPEZLWKLQKRUODWHUEXWLQWKHDEVHQFHRIO\VVDYLUXVDQWLJHQVLQWKHLU EUDLQV 7KLV XVXDOO\ UHVXOWV IURP EDFWHULDO RU RWKHU YLUDO FRQWDPLQDWLRQ RI WKH LQRFXOXP 7R UHPRYH EDFWHULDO FRQWDPLQDWLRQ WKH LQRFXOXP FDQ EH ĺOWHUHG WKURXJKDPĺOWHUDWWDFKHGWRDV\ULQJH6KRXOGYLUDOFRQWDPLQDWLRQRFFXU there is no realistic way to separate an unwanted virus from a lyssavirus present LQWKHVDPSOH7KH}H[FHSWLRQVLQFOXGHRQO\VRPHPRXVHYLUXVHVZKLFKDUHQRW SDWKRJHQLFIRURWKHUDQLPDOV,QVXFKDFDVHWKHLQRFXOXPFDQEHLQMHFWHGLQWUDFUD nially or intramuscularly into other mammalian species (for example, Syrian hams WHUV ZKHUHWKHPXULQHYLUXVVKRXOGQRWUHSOLFDWHEXWWKHO\VVDYLUXVZLOO Virus isolation in animals Laboratory techniques in rabies Fifth edition Part 2. Detection of virus If a lyssavirus cannot be isolated from a sample that demonstrates abundant SUHVHQFHRIYLUDODQWLJHQVRU51$WKLVFDQUHVXOWIURPVLJQLĺFDQWGHJUDGDWLRQRI infectious virus (for example, due to inappropriate storage) or from high titres of YLUXVQHXWUDOL]LQJDQWLERGLHVLQWKHVSHFLPHQ,IDVDPSOHLVGHJUDGHGDKLJKHU FRQFHQWUDWLRQ RI WKH LQRFXOXP FDQ EH XVHG IRU H[DPSOH WLVVXH VXVSHQ VLRQ DQGQHZERUQ DJHGtGD\V PLFHLQRFXODWHGWRLQFUHDVHWKHVHQVLWLYLW\RI WKH0,7,IYLUXVQHXWUDOL]LQJDQWLERGLHVDUHSUHVHQWLQWKHVDPSOHVHYHUDOVHULDO IROGGLOXWLRQVRIWKHLQRFXOXPFDQLPSURYHWKHFKDQFHVRIYLUXVLVRODWLRQ7KH dilutions should be made promptly after preparation of the original inoculum, and cold diluent should be used at each step to decrease binding of antibodies to the YLUXVUHOHDVHGIURPWKHWLVVXHFHOOVGXULQJWKHKRPRJHQL]DWLRQSURFHVV Virus titration Quantitation of virus via determination of an infectious or lethal dose can EHSHUIRUPHGXVLQJWLWUDWLRQLQFHOOFXOWXUHRULQDQLPDOPRGHOVLQFOXGLQJPLFH 7KH}WLWUHVREWDLQHGLQDQLPDOPRGHOVDQGLQFHOOFXOWXUHXVXDOO\FRUUHODWHDOWKRXJK the titres of infectious virus may not necessarily correlate with antigenic load deter mined by ELISA or with the number of genome copies determined by quantitative 573&5&HOOFXOWXUHRIIHUVVHYHUDODGYDQWDJHVIRUYLUXVWLWUDWLRQFRPSDUHGZLWK DQLPDOPRGHOV7KHWHVWLQFHOOFXOWXUHLVPRUHUDSLGDQGHYHQWXDOO\LVOHVVFRVWO\ KRZHYHULWUHTXLUHVFHOOFXOWXUHFDSDFLW\0RXVHLQRFXODWLRQFDQEHSHUIRUPHGLQD PRUHEDVLF%6/ODERUDWRU\ZLWKDYLYDULXP2WKHUH[DPSOHVZKHUHGHWHUPLQDWLRQ of lethal doses in animals is preferable include the evaluation of susceptibility in vivo and pathogenesis studies, potency tests for biologicals, and comparison of YLUXOHQFHYLDLQWUDFUDQLDODQGSHULSKHUDOURXWHV 6HYHUDOVHULDOGLOXWLRQVRIYLUDOLQRFXOXPDUHXVHGIRUDWLWUDWLRQ0RVWFRPPRQO\ IROGGLOXWLRQVDUHXVHGIRUGHWHUPLQDWLRQRIYLUXVWLWUHLQPLFHDOWKRXJKRWKHU GLOXWLRQIDFWRUV IRUH[DPSOHIROGIROGIROG FDQEHXVHGDVZHOO7KHOHVV WKHGLOXWLRQIDFWRUWKHPRUHSUHFLVHLVWKHWLWUHGHWHUPLQHG+RZHYHUDGHFUHDVH RIWKHGLOXWLRQIDFWRUZLOOLQFUHDVHWKHQXPEHURIDQLPDOVXVHG)RUH[DPSOHVHYHQ IROGGLOXWLRQV VHYHQJURXSVRIPLFH DUHXVXDOO\VXIĺFLHQWWRGHWHUPLQHO\VVD YLUXVWLWUHVLQPRVWĺHOGVDPSOHV7RFRYHUWKHVDPHWLWUHUDQJHYLDIROGGLOXWLRQV JURXSVRIPLFHDUHQHHGHG(YHQJURXSVRIPLFHZRXOGEHQHHGHGWRFRYHU WKLVUDQJHYLDIROGGLOXWLRQV7KHVL]HRIWKHJURXSLQRFXODWHGZLWKRQHGLOXWLRQ FDQ DOVR YDU\ 8VXDOO\ t PLFH SHU JURXS DUH XVHG IRU JHQHULF WLWUDWLRQV EXW JURXSVDVODUJHDVRUHYHQPLFHFDQEHXVHGIRUVXFKLPSRUWDQWSURFHGXUHV DVGHWHUPLQDWLRQRIYDFFLQHSRWHQF\WHVWIRUVWDWLVWLFDOFRPSDULVRQV $OO GLOXWLRQV DUH LQRFXODWHG LQ PLFH IROORZLQJ WKH VWDQGDUG 0,7 SURFHGXUH GHVFULEHGDERYH,WLVSUHIHUDEOHWKDWWKHLQRFXODWLRQLVPDGHE\RQHRSHUDWRUWR LPSURYHFRPSDUDELOLW\RIWKHUHVXOWVEHWZHHQJURXSV7KHREVHUYDWLRQSHULRGIRU PLFHLQRFXODWHGLQWUDFUDQLDOO\FDQXVXDOO\EHUHGXFHGWRGD\V/RQJHULQFXED WLRQSHULRGVDUHUDUHDQGHYHQLItPLFHDUHPLVVHGEHFDXVHRIWKHREVHUYDWLRQ VKRUWHQLQJWKH\ZLOOQRWDIIHFWWKHREWDLQHGWLWUHVLJQLĺFDQWO\+RZHYHUIRUPLFH LQRFXODWHGSHULSKHUDOO\WKHREVHUYDWLRQVKRXOGEHDWOHDVWGD\V SUHIHUDEOH GD\V EHFDXVHORQJHULQFXEDWLRQSHULRGVLQVXFKDQLPDOVRFFXUPRUHIUHTXHQWO\ $IWHUFRPSOHWLRQRIWKHH[SHULPHQWWKHPLFHIURPWKHHQGSRLQWGLOXWLRQWKDW caused mortality and from at least one dilution above are examined for the SUHVHQFHRIYLUDODQWLJHQVLQWKHLUEUDLQV7KHWLWUHVDUHFDOFXODWHGYLDWKH5HHG 0XHQFKRU6SHDUPDQt.ÁUEHUPHWKRGVDVGHVFULEHGHOVHZKHUH Virus isolation in animals Laboratory techniques in rabies Fifth edition Part 2. Detection of virus References .RSURZVNL + 7KH PRXVH LQRFXODWLRQ WHVW ,Q 0HVOLQ ); .DSODQ 00 .RSURZVNL}+HGLWRUV/DERUDWRU\WHFKQLTXHVLQUDELHVWKHGLWLRQ*HQHYD :RUOG +HDOWK2UJDQL]DWLRQ t 0DUNRWWHU:.X]PLQ,95XSSUHFKW&(1HO/+/DJRVEDWYLUXVYLUXOHQFHLQ PLFH LQRFXODWHG E\ WKH SHULSKHUDO URXWH (SLGHPLRO ,QIHFW t GRL 6 )DEHU 0 /L - .HDQ 5% +RRSHU '& $OXJXSDOOL .5 'LHW]VFKROG % (IIHF tive preexposure and postexposure prophylaxis of rabies with a highly atte QXDWHGUHFRPELQDQWUDELHVYLUXV3URF1DWO$FDG6FL86$ t GRL SQDV %RXUK\+5ROOLQ3(9LQFHQW-6XUHDX3&RPSDUDWLYHĺHOGHYDOXDWLRQRIWKH ĻXRUHVFHQWDQWLERG\WHVWYLUXVLVRODWLRQIURPWLVVXHFXOWXUHDQGHQ]\PHLPPX QRGLDJQRVLV IRU UDSLG ODERUDWRU\ GLDJQRVLV RI UDELHV - &OLQ 0LFURELRO t KWWSV SGIVVHPDQWLFVFKRODURUJIHG FIEDEDEDHHE GDIHHIFDFSGIDFFHVVHG0D\ 6KRSH5(0XUSK\)$+DUULVRQ$.&DXVH\25.HPS*(6LPSVRQ',HWDO 7ZR$IULFDQ YLUXVHV VHURORJLFDOO\ DQG PRUSKRORJLFDOO\ UHODWHG WR UDELHV YLUXV - 9LURO t30,' *DUJLXOR6*UHFR$*UDPDQ]LQL0(VSRVLWR6$IIXVR$%UXQHWWL$HWDO0LFH anesthesia, analgesia, and care, Part I: anesthetic considerations in preclinical UHVHDUFK,/$5- (t GRL LODU 7D\ORU%-2UU6$&KDSPDQ-/)LVKHU'(%H\RQGXVHGDWLQJRIH[WHPSR UDQHRXVO\FRPSRXQGHGNHWDPLQHDFHSURPD]LQHDQG[\OD]LQH VDIHW\VWDELOLW\DQG HIĺFDF\RYHUWLPH-$P$VVRF/DE$QLP6FL 30,' $90$JXLGHOLQHVIRUWKHHXWKDQDVLDRIDQLPDOV HGLWLRQ6FKDXPEXUJ ,/ $PHULFDQ9HWHULQDU\0HGLFDO$VVRFLDWLRQ Virus isolation in animals Laboratory techniques in rabies Fifth edition Part 2. Detection of virus Chapter 9 Virus isolation in cell culture: the rabies tissue culture infection test Introduction 7KHDLPRIWKHUDELHVWLVVXHFXOWXUHLQIHFWLRQWHVW 57&,7 LVWRGHWHFWLQYLWUR XVLQJFHOOFXOWXUHWHFKQLTXHVWKHSUHVHQFHRILQIHFWLRXVUDELHVYLUXV 5$%9 RU RWKHUO\VVDYLUXVHVLQWLVVXHVXVSHQVLRQVRULQOLTXLGELRORJLFDOVDPSOHV7KH57&,7 QRZZHOOGHYHORSHGDQGVWDQGDUGL]HGLVZLGHO\LPSOHPHQWHGDQGLVFRQVLGHUHG using brain biopsy specimens as a conventional routine test for the postmortem GLDJQRVLVRIUDELHVLQDQLPDOVDQGKXPDQVDQGDFWLQJDVDFRQĺUPDWRU\PHWKRGWR WKHGLUHFWĻXRUHVFHQWDQWLERG\WHVW ')$7 ZKLFKLVWKHJROGVWDQGDUGWHFKQLTXH Application of this test can also be extended to the analysis of other types of VDPSOHLQFOXGLQJERG\ĻXLGVVXFKDVVDOLYDDQGFHUHEURVSLQDOĻXLG &6) LQ}WKH GLDJQRVLVRIKXPDQUDELHVERWKSRVWPRUWHPRUDQWHPRUWHP ,QLWLDOO\ WKH ĺUVW DWWHPSWV WR LVRODWH 5$%9 LQ FHOO FXOWXUH IURP VXVSHFWHG VDPSOHV ZHUH SHUIRUPHG LQ WKH V XVLQJ YDULRXV FHOO OLQHV LQFOXGLQJ EDE\ KDPVWHU NLGQH\ %+. FKLFN HPEU\RUHODWHG RU QHXUREODVWRPD FHOOV (1–6) 7KH}ODWWHUZHUHGHPRQVWUDWHGWREHPRUHVXVFHSWLEOHWRĺHOGLVRODWHVRI5$%9 than other cell lines tested, probably due to their neuronal origin (7, 8)9LUXV}VROD WLRQLQQHXUREODVWRPDFHOOFXOWXUHZDVIXUWKHUGHPRQVWUDWHGWREHDWOHDVWDVHIĺ cient as animal inoculation, especially for small quantities of virus, and was rapidly XVHGURXWLQHO\WRUHSODFH5$%9LVRODWLRQLQQHZERUQPLFHLQGLIIHUHQWODERUDWRULHV LQWKHHDUO\ V(9, 10) 9LUDO LQIHFWLRQ LQ FHOOV SURGXFHV VSHFLĺF LQFOXVLRQV GHWHFWDEOH JHQHUDOO\ t} KRXUV DIWHU LQIHFWLRQ UDQJLQJ DV HDUO\ DV t K XS WR GD\V E\ GLUHFW LPPXQRĻXRUHVFHQFH DIWHU FHOO ĺ[DWLRQ DQG VWDLQLQJ ZLWK D VSHFLĺF DQWLUDELHV ĻXRUHVFHQWFRQMXJDWHGDQWLERG\(1–4, 11) )LJ 7KHUHIRUHYLUXVLVRODWLRQLQFHOO FXOWXUH SURYLGHV PRUH UDSLG UHVXOWV WKDQ LQRFXODWLRQ LQ DQLPDOV t GD\V RU ORQJHU DQGUHPDLQVOHVVH[SHQVLYH7KLVWHFKQLTXHLVDOVRPRUHHWKLFDOEHFDXVH it replaces the use of animals (12)7KXV57&,7UHSUHVHQWVDUHIHUHQFHWHFKQLTXH IRULVRODWLRQRIO\VVDYLUXVHVDQGVKRXOGUHSODFHWKHPRXVHLQRFXODWLRQWHVW 0,7 whenever possible (13, 14) 9LUXVLVRODWLRQLVJHQHUDOO\SHUIRUPHGDWWKHSRVWPRUWHPGLDJQRVLVRIUDELHV RQ EUDLQ VSHFLPHQV WR FRQĺUP WKH UHVXOWV REWDLQHG E\ WKH GHWHFWLRQ RI O\VVD YLUXVDQWLJHQVXVLQJWKH')$7HVSHFLDOO\ZKHQWKHUHVXOWVDUHQHJDWLYHRUXQFHU tain (13, 14) &RPSDUHG ZLWK WKH ')$7 WKLV WHFKQLTXH KDV GHPRQVWUDWHG DQ RYHUDOOFRQFRUGDQFHRI ZLWKDVSHFLĺFLW\RIDQGDVHQVLWLYLW\RI t}(11)+RZHYHUDPDMRUOLPLWDWLRQRIWKLVWHFKQLTXHLVWKHSUHVHUYDWLRQ of the infectivity in the submitted samples, as suboptimal conditions of samples (such as decomposed specimens) could induce false–negative results (15) Virus isolation in cell culture Laboratory techniques in rabies Fifth edition Part 2. Detection of virus 5HSURGXFHGZLWKSHUPLVVLRQIURP 'DFKHX[/%RXUK\+9LUXVLVRODWLRQLQFHOOFXOWXUH WKH UDELHVWLVVXHFXOWXUHLQIHFWLRQWHVW,Q 5XSSUHFKW&1DJDUDMDQ7HGLWRUV&XUUHQWODERUDWRU\ WHFKQLTXHVLQUDELHVGLDJQRVLVUHVHDUFKDQGSUHYHQWLRQ9RO1HZ )LJ 6FKHPDWLFUHSUHVHQWDWLRQRIWKH57&,7 UDELHVWLVVXHFXOWXUHLQIHFWLRQWHVW SURWRFROEDVHGRQWKHXVHRIDUDELHVDQWL QXFOHRFDSVLG),7& ĻXRUHVFHLQLVRWKLRF\DQDWH FRQMXJDWHGDQWLERG\ RIUDEELWRULJLQ 7KH57&,7FDQDOVREHDSSOLHGWRWKHDQWHPRUWHP DQGSRVWPRUWHP GLDJQRVLV XVLQJELRORJLFDOĻXLGVVXFKDV&6)ODFULPDOĻXLGRUVDOLYD+RZHYHUWKHVHQVLWLYLW\ of the test will vary according to the intermittence of viral excretion and the level of YLUDOORDGWKHQXPEHURIFRQVHFXWLYHFHOOSDVVDJHVSHUIRUPHGEHIRUHĺ[DWLRQRI WKHFHOOVDQGWKHLPPXQRORJLFDOVWDWXVRIWKHSDWLHQWDVWKHSUHVHQFHRIVSHFLĺF 5$%9DQWLERGLHVHVSHFLDOO\WKRVHZLWKQHXWUDOL]LQJDFWLYLW\PD\QHJDWLYHO\DIIHFW the rate of positivity (14)7KHTXDOLW\RIWKRVHVDPSOHVVLPLODUO\WRRWKHUGLDJQRVWLF tests, is of utmost importance and the samples should therefore be frozen imme diately after collection, preferentially in dry tubes (medium containing preserva WLYHVVKRXOGQRWEHXVHG In addition to diagnosis, virus isolation represents a necessary step to amplify DQ\ĺHOGLVRODWHIRULWVIXUWKHUFKDUDFWHUL]DWLRQ Virus isolation in cell culture Laboratory techniques in rabies Fifth edition Part 2. Detection of virus Protocol 7KH5&7,7WHFKQLTXHFDQEHSHUIRUPHGXVLQJHLWKHUPXOWLSOHFKDPEHUVOLGHV HJFKDPEHUVOLGHV>VXFKDV/DE7HN@ RUPLFURSODWHV HJ ZHOOSODWHV Reagents and equipment Reagents sFHOOFXOWXUHPHGLXP'0(0 VHHAnnex 1 for composition) • suspension medium (grinding medium, see Annex 3 for composition) sQHXWUDO3%6 sDFHWRQH GLOXWHGLQ3%6 • trypsin (sterility of each new batch should be tested after inoculation of brain heart infusion broth) s'($(GH[WUDQ JLYHQDWDĺQDOFRQFHQWUDWLRQRISJP/ s5HDJHQWV IRU ')$7 LQFOXGLQJ VSHFLĺF DQWLUDELHV ĻXRUHVFHQW FRQMXJDWHG antibody (either polyclonal or monoclonal antibodies, generally labelled with WKHĻXRUHVFHLQLVRWKLRF\DQDWH ),7& ĻXRURSKRUH FRPPHUFLDOO\DYDLODEOHRU validated homemade antibodies), used at the recommended dilution with DSSURSULDWHGLOXHQWFRXQWHUVWDLQLQJ (YDQV%OXHJHQHUDOO\DWGLOXWLRQ DQGPRXQWLQJPHGLXP JO\FHUROGLOXWHGLQ3%6 • Supernatant of homogenized, controlled infected and naive brain tissues, XVHG DV SRVLWLYH DQG QHJDWLYH FRQWUROV UHVSHFWLYHO\ HJ PRXVH EUDLQ WLVVXHV Equipment s %URHNW\SH JODVV KRPRJHQL]HUV RU JODVV URGV DGDSWHG WR WKH SODVWLF FHQWULIXJHWXEHV P/WXEHV s &ODVVELRVDIHW\FDELQHWIRUFHOOFXOWXUHDQGPDQLSXODWLRQRI5$%9XVLQJ standard biosafety measures s HLJKWFKDPEHUVOLGHV HJ/DE7HNJODVVRU3HUPDQR[VOLGHV RUPLFURSODWHV HJ ZHOOSODWHV ZLWKĻDWERWWRPFOHDUFRORXUDGDSWHGWRDGKHUHQWFHOO culture s GU\LQFXEDWRUXVHGDW&& • humid, CO controlled incubator used for routine cell maintenance and for WKH57&,7SURFHGXUHXVHGDW&2DQG&& • refrigerated centrifuge s ĻXRUHVFHQFHPLFURVFRSHZLWKDSSURSULDWHGĺOWHUVIRUWKHZDYHOHQJWKRIWKH ĻXRUHVFHQWG\H IRU),7& PD[LPXPH[FLWDWLRQZDYHOHQJWKDW QPDQG HPLVVLRQ ZDYHOHQJWK DW QP ,Q SDUWLFXODUWKH PLFURVFRSH VKRXOG EH HTXLSSHGZLWKDORQJIRFDOOHQVLIXVLQJPLFURSODWHV HJ ZHOOSODWHV WR DOORZWKHSODWHWREHUHDGLQDQLQYHUWHGSRVLWLRQ7KLVVWHSFRXOGEHVLPSOL ĺHGXVLQJGLUHFWO\DQLQYHUWHGĻXRUHVFHQFHPLFURVFRSH s DSSURSULDWHYLUXFLGDOVROXWLRQ Virus isolation in cell culture Laboratory techniques in rabies Fifth edition Part 2. Detection of virus Choice and maintenance of neuroblastoma cell cultures s 0XULQH QHXUREODVWRPD FHOOV VXFK DV 1HXURD $7&& &&/ KWWSV ZZZOJFVWDQGDUGVDWFFRUJSURGXFWVDOO&&/DVS["JHRBFRXQWU\ IU), VKRXOGEHXVHGGXHWRWKHLUKLJKVHQVLWLYLW\WR5$%9DQGRWKHUO\VVDYLUXV LQIHFWLRQ s 0DLQWDLQ WKH QHXUREODVWRPD FHOOV LQ FP SODVWLF ĻDVNV LQ FHOO FXOWXUH PHGLXP $QQH[ DW&&LQD&2KXPLGLĺHGLQFXEDWRU 7U\SVLQL]HWKHFHOOVWZLFHDZHHN'HWDLOVUHJDUGLQJPDLQWHQDQFHDQGFRQVHU vation of murine neuroblastoma cells are described in Annex 2 Preparation of the cell suspension 7U\SVLQL]HRQHĻDVNRIVXEFRQĻXHQWFHOOVIRUWKHFHOOVXVSHQVLRQSUHSDUDWLRQ DQGUHVXVSHQGWKHFHOOVDWDFRQFHQWUDWLRQRI´FHOOVP/ IRUFKDPEHU VOLGHV RUDWDFRQFHQWUDWLRQRI´FHOOVP/ IRU ZHOOPLFURSODWHV LQ '0(0LQDP/WXEH 7KLVSUHSDUDWLRQFDQEHNHSWXQWLOGD\VDW& Preparation of the inoculum For brain samples 3UHSDUHDVXVSHQVLRQ PDVVYRO RIWKHEUDLQVSHFLPHQLQWKHVXVSHQ sion medium (grinding medium, Annex 3 XVLQJ%URHNW\SHJODVVKRPRJH QL]HUVRUJODVVURGVDGDSWHGWRSODVWLFFHQWULIXJHWXEHV HJP/WXEH Different brain tissues should be selected after autopsy or necropsy, inclu GLQJWKHEUDLQVWHPFRUWH[FHUHEHOOXPDQGKLSSRFDPSXV ZKHQDYDLODEOH 7KHVXVSHQVLRQPXVWEHSHUIRUPHGLQDFODVVELRVDIHW\FDELQHW &ODULI\WKHVXVSHQVLRQE\FHQWULIXJDWLRQDW´gIRUPLQDW& For liquid samples 6DOLYDVDPSOHVPD\EHGLOXWHG YROYRO LQVXVSHQVLRQPHGLXPDVZHOODV ODFULPDOVDPSOHV6WHULOH&6)VSHFLPHQVPD\EHLQRFXODWHGXQGLOXWHG Inoculation Inoculation using 8-chamber slides (Fig. 9.2) 'LVWULEXWH/RIWKHQHXUREODVWRPDFHOOVXVSHQVLRQ ´FHOOVP/ SHU FKDPEHU FHOOVFKDPEHU WRUHDFKDQFRQĻXHQWFHOOPRQROD\HU $GG/RIHDFKFODULĺHGEUDLQKRPRJHQDWHLQRQHFKDPEHUSHUVDPSOHDQG PL[JHQWO\ XSDQGGRZQSLSHWWLQJ EHLQJFDUHIXOQRWWRJHQHUDWHDHURVROV ,QFOXGHDWOHDVWRQHSRVLWLYHFRQWURO ĺ[HGRUĺHOGLVRODWH DQGRQHQHJDWLYH FRQWURO FRQĺUPHG QHJDWLYH EUDLQ IRU HDFK VHULHV RI VOLGHV SUHIHUDEO\ RQ D separate slide or on the same slide as the samples, but using a well as distant DVSRVVLEOHIURPWKHZHOOVXVHGIRUWKHWHVWHGVDPSOHV Virus isolation in cell culture Laboratory techniques in rabies Fifth edition Part 2. Detection of virus ,QFXEDWH IRU t K DW & & LQ D &2 KXPLGLĺHG LQFX EDWRU$}ORQJHULQFXEDWLRQSHULRGPD\EHUHTXLUHG HJKGXULQJZHHNHQGV LQ ZKLFK FDVH LW LV VXJJHVWHG WR UHGXFH WKH QXPEHU RI FHOOV FHOOV FKDPEHU Inoculation using microplates (e.g. 96-well plates) (adapted from 7, 13) 'LVWULEXWH/RIWKHQHXUREODVWRPDFHOOVXVSHQVLRQ ´FHOOVP/ SHU ZHOO FHOOVZHOO LQIRXUZHOOVRID ZHOOSODWHWRUHDFKDQFRQĻXHQW FHOOPRQROD\HU $GG/RIHDFKFODULĺHGEUDLQKRPRJHQDWHLQHDFKRIWKHIRXUZHOOVDQG PL[JHQWO\ XSDQGGRZQSLSHWWLQJ ,QFOXGHDWOHDVWRQHSRVLWLYH ĺ[HGRUĺHOG LVRODWH DQGRQHQHJDWLYH FRQĺUPHGQHJDWLYHEUDLQ FRQWUROIRUHDFKVHULHVRI SODWHV ,QFXEDWHIRUtGD\VDW&&LQD&2KXPLGLĺHGLQFXEDWRU Changing of cell medium can be eventually performed during this incubation SHULRG %\FRXUWHV\RI/DXUHQW'DFKHX[DQG+HUYÆ%RXUK\ 3DVWHXU,QVWLWXWHRI)UDQFH )LJ ,OOXVWUDWLRQVRIZHOOFKDPEHUVOLGHV $ 'HSRVLWLQJWKHFHOOPHGLXPLQWRWKHZHOOFKDPEHUVOLGHV % 5HPRYLQJWKHZHOO LQFXEDWLQJFKDPEHUV & 6OLGHVZLWKDQGZLWKRXWZHOOLQFXEDWLQJFKDPEHUV )L[DWLRQRIFHOOVDQGYLUXVGHWHFWLRQ )L[DWLRQXVLQJFKDPEHUVOLGHV HJ/DE7HN )LJ 'LVFDUGFDUHIXOO\WKHPHGLXPIURPHDFKFKDPEHULQDYDOLGDWHGYLUXFLGDOVROXWLRQ )L[WKHFHOOVZLWKFROGDFHWRQHIRUPLQDW& 'U\WKHVOLGHVDWURRPWHPSHUDWXUHIRUtPLQ 6WDLQ ZLWK WKH VSHFLĺF DQWLUDELHV ),7&FRQMXJDWHG DQWLERG\ / SHU FKDPEHUXVLQJDQDSSURSULDWHGLOXWLRQZLWKDGGLWLRQRIFRXQWHUVWDLQLQJ GXULQJ PLQDW&&LQDKXPLGLĺHGLQFXEDWRU 5HPRYHWKHSODVWLFFKDPEHUVpIUDPH 5LQVHWKHVOLGHVLQWZRVXFFHVVLYHEDWKVRISKRVSKDWHEXIIHUHGVDOLQH 3%6 7KLVZDVKLQJPXVWEHYHU\JHQWOHWRSUHYHQWUHPRYDORIWKHFHOOV &RYHUZLWKPRXQWLQJPHGLXP JO\FHUROLQ3%6 DQGFRYHUVOLS 5HDGXVLQJWKHĻXRUHVFHQFHPLFURVFRSH Virus isolation in cell culture Laboratory techniques in rabies Fifth edition Part 2. Detection of virus Inoculation using microplates (e.g. 96-well plate) (adapted from 7, 13) 'LVFDUGFDUHIXOO\WKHPHGLXPIURPHDFKZHOOLQDQDSSURSULDWHYLUXFLGDOVROX WLRQDQGSHUIRUPRQHZDVKZLWK3%6 )L[WKHFHOOVZLWKFROGDFHWRQHIRUPLQDW& 'U\WKHVOLGHVDWURRPWHPSHUDWXUHIRUtPLQ 6WDLQZLWKWKHVSHFLĺFDQWLUDELHV),7&FRQMXJDWHGDQWLERG\ /SHUZHOO XVLQJDQDSSURSULDWHGLOXWLRQZLWKDGGLWLRQRIFRXQWHUVWDLQLQJ IRUPLQDW }&&LQDKXPLGLĺHGLQFXEDWRU :DVKWZLFHWKHZHOOVZLWK3%67KLVZDVKLQJPXVWEHYHU\JHQWOHWRSUHYHQW UHPRYDORIWKHFHOOV $GGDGURSRIPRXQWLQJPHGLXPLQHDFKZHOO JO\FHUROLQ3%6 5HDGXVLQJWKHĻXRUHVFHQFHPLFURVFRSH Interpretation of results 7KH SUHVHQFH RI EULJKW JUHHQ ĻXRUHVFHQW LQFOXVLRQV LQ WKH F\WRSODVP RI WKH FHOOVFDQEHREVHUYHGXVLQJWKHĻXRUHVFHQFHPLFURVFRSH )LJ 2SWLPDOĺQDO PDJQLĺFDWLRQZLWKFKDPEHUVVOLGHVLV[DQGZLWKPLFURSODWHV HJ ZHOO SODWH DĺQDOPDJQLĺFDWLRQRIt[LVDGDSWHGXVLQJDPLFURVFRSHHTXLSSHG by a longer focal lens to read the plates in an inverted position (this step can be VLPSOLĺHGXVLQJDQLQYHUWHGĻXRUHVFHQFHPLFURVFRSH XWHRI)UDQFH %\FRXUWHV\RI/DXUHQW'DFKHX[DQG+HUYÆ%RXUK\3DVWHXU,QVWLW )LJ ([DPSOHVRIPLFURVFRSLFREVHUYDWLRQV PDJQLĺFDWLRQ[ RI57&,7VOLGHVDIWHUODEHOOLQJZLWKDVSHFLĺFDQWLUDELHV),7& FRQMXJDWHGDQWLERG\ $ 3RVLWLYH SUHVHQFHRILQIHFWLRQ VOLGHZLWKWKHREVHUYDWLRQRIEULJKWJUHHQĻXRUHVFHQWLQFOXVLRQVLQWKHF\WRSODVPLQGLFDWLQJ5$%9 infection % 1HJDWLYH DEVHQFHRILQIHFWLRQ VOLGHZLWKFHOOVREVHUYHGLQUHGDIWHUFRXQWHUVWDLQLQJZLWK(YDQV%OXH Virus isolation in cell culture Laboratory techniques in rabies Fifth edition Part 2. Detection of virus Discussion 7KH57&,7UHSUHVHQWVDWHFKQLTXHRIUHIHUHQFHIRUO\VVDYLUXVLVRODWLRQ(13, 14) 7KLV FKDSWHU GHVFULEHV D YDOLGDWHG SURWRFRO RI 57&,7 XVLQJ FKDPEHUV VOLGHV HJ/DE7HN ZKLFKKDVEHHQGHPRQVWUDWHGWRSURYLGHUHOLDEOHDQGUDSLGUHVXOWV DVIDVWDVtKDIWHULQRFXODWLRQ(11), as well as the initial method performed in microplates (7, 13) Whatever the technique used, suboptimal sample conditions, such as decom SRVHGEUDLQDUHDPDMRUOLPLWDWLRQWKDWFRXOGLQGXFHDORVVRILQIHFWLYLW\DQG\LHOG false–negative results (15) &\WRWR[LFLW\ FDQ DOVR UHVXOW IURP VXFK VDPSOHV GXH WRWKHSUHVHQFHRILQIHFWLRQLQKLELWLQJIDFWRUVLQEUDLQVXVSHQVLRQVOLPLWLQJWKH robustness of the test (10, 11)+RZHYHUWKHDGGLWLRQRIDQWLELRWLFVUHGXFWLRQRIWKH WLPHEHIRUHFKDQJLQJPHGLDRUGLOXWLRQRIVDPSOHVFDQUHGXFHWKLVF\WRWR[LFLW\ 9DULDWLRQVLQWKLVWHFKQLTXHH[LVWDQGPDLQO\LQFOXGHWKHW\SHRIPDWHULDOXVHG IRUFHOOFXOWXUH ZLWKRWKHUPXOWLZHOOSODVWLFSODWHVRUJODVVFRYHUVOLSV DVZHOO as the process of inoculation with incubation time, further passages and use of FHOOSHUPHDELOLW\DJHQWV/RQJHULQFXEDWLRQSHULRGV tGD\V FDQEHURXWLQHO\ FRQGXFWHGHVSHFLDOO\IRUQHJDWLYHVDPSOHVWRLQFUHDVHVHQVLWLYLW\5HSODFHPHQW of cell medium during this longer incubation period is not necessary but can EHGRQH,QDGGLWLRQVXFFHVVLYHSDVVDJHVFDQDOVREHSHUIRUPHG XSWRWKUHH SDVVDJHVPD\EHFRQVLGHUHGWRLQFUHDVHVHQVLWLYLW\ DQGWKHVXSHUQDWDQWVNHSW IRUIXUWKHUSDVVDJHVLIQHFHVVDU\,QWKLVFDVHWKHWLPHUHTXLUHGWRĺQDOL]HWKH assay should be compatible with the limited delay available in some cases so as to inform the physician in charge of the decision regarding prophylaxis for the H[SRVHG SDWLHQWV 3HUPHDELOLW\ DJHQWV VXFK DV GLHWK\ODPLQRHWK\O '($( GH[ WUDQFDQEHDGGHGWRWKHFHOOVXVSHQVLRQLPPHGLDWHO\EHIRUHXVHWRJLYHDĺQDO FRQFHQWUDWLRQRISJP/ 7KHXVHRIFHOOOLQHVRWKHUWKDQQHXUREODVWRPDFHOOV has been suggested but is not recommended (13, 14)&HOOFXOWXUHWHVWVDQGWKHLU YDULDWLRQVVKRXOGEHIXOO\YDOLGDWHGEHIRUHXVH $OWKRXJKWKH57&,7LVUHODWLYHO\HDV\WRSHUIRUPVRPHFULWLFDOSDUDPHWHUVPXVW EHHPSKDVL]HG,PSOHPHQWDWLRQRIWKHWHVWUHTXLUHVWKDWVWULFWVWDQGDUGRSHUD ting procedures for cell culture be followed, with appropriate equipment and SURĺFLHQF\RIVWDII,QSDUDOOHOEHFDXVHGHWHFWLRQLVEDVHGRQWKHGLUHFWLPPX QRĻXRUHVFHQFHRIYLUDODQWLJHQVVLPLODUWHFKQLFDOUHFRPPHQGDWLRQVUHODWHGWRWKH ')$7PXVWDOVREHIROORZHGVXFKDVWKHFKRLFHDQGYDOLGDWLRQRIWKHVSHFLĺF DQWLUDELHVĻXRUHVFHQWFRQMXJDWHGDQWLERG\GLOXWLRQDQGLQFXEDWLRQFRQGLWLRQV washing steps and mounting medium (13, 14, 16),QSDUWLFXODUWKHĺQDOUHDGLQJVWHS is of utmost importance and must be performed by trained operators (up to two GLIIHUHQWUHDGHUVSHUVOLGHRUPLFURSODWH XVLQJDGDSWHGPDJQLĺFDWLRQDVZHOODV DUHJXODUO\FRQWUROOHGDQGZHOOPDLQWDLQHGĻXRUHVFHQFHPLFURVFRSH3DUWLFLSDWLRQ RIWKHODERUDWRU\WRDSURĺFLHQF\WHVWLVVWURQJO\VXJJHVWHGIRUWKLVWHFKQLTXH(17) Virus isolation in cell culture Laboratory techniques in rabies Fifth edition Part 2. 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Detection of virus 0F(OKLQQH\ /0 0DUVWRQ '$ %URRNHV 60 )RRNV $5 (IIHFWV RI FDUFDVH GHFRPSRVLWLRQRQUDELHVYLUXVLQIHFWLYLW\DQGGHWHFWLRQ-9LURO0HWKRGV t 'HDQ'-$EHOVHWK0.$WDQDVLX37KHĻXRUHVFHQWDQWLERG\WHVW,Q 0HVOLQ); .DSODQ00.RSURZVNL+HGLWRUV/DERUDWRU\WHFKQLTXHVLQUDELHVWKHGLWLRQ *HQHYD :RUOG+HDOWK2UJDQL]DWLRQ t 5REDUGHW ( 3LFDUG0H\HU ( $QGULHX 6 6HUYDW $ &OLTXHW ) ,QWHUQDWLRQDO interlaboratory trials on rabies diagnosis: an overview of results and variation in UHIHUHQFHGLDJQRVLVWHFKQLTXHV ĻXRUHVFHQWDQWLERG\WHVWUDELHVWLVVXHFXOWXUH LQIHFWLRQWHVWPRXVHLQRFXODWLRQWHVW DQGPROHFXODUELRORJ\WHFKQLTXHV-9LURO 0HWKRGV t 3RXOLRWW&'XSXLV0$SSOHU.%UXQW65XGG5'DYLV$6XVFHSWLELOLW\RI QHXUREODVWRPDFHOOVWRUDELHVYLUXVPD\EHDIIHFWHGE\SDVVDJHQXPEHU-}9LURO 0HWKRGV t Virus isolation in cell culture Laboratory techniques in rabies Fifth edition Part 2. Detection of virus Annex 1. Composition of cell culture medium DMEM10 s 'XOEHFFRpVPRGLĺHG(DJOHpVPHGLXP '0(0 ZLWKKLJKJOXFRVH PJ/ VXSSOHPHQWHGZLWK/DODQ\O/JOXWDPLQH *OXWD0$; PJ/ DQGS\UXYDWH PJ/ VXSSOHPHQWHGPHGLXPFRPPHUFLDOO\DYDLODEOH s RIKHDWLQDFWLYDWHGIHWDOFDOIVHUXP )&6 s 2SWLRQDOO\ DSSURSULDWH DQWLELRWLFV VXFK DV JHQWDPLFLQ DW ĺQDO FRQFHQWUDWLRQ }JP/ • Each new reconstitution of cell culture medium should be tested for sterility (for example, after inoculation and incubation of a small volume in brain heart LQIXVLRQEURWK 7KHTXDOLW\RIQHZEDWFKHVRI)&6VKRXOGDOVREHWHVWHGLQ comparison with previous validated batches (including the evaluation of cell PXOWLSOLFDWLRQDQGWKHSUHVHQFHRI5$%9QHXWUDOL]LQJDFWLYLW\ Annex 2. 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Detection of virus Chapter 10 Transmission electron microscopy in rabies diagnosis, ultrastructural studies and research Introduction (OHFWURQPLFURVFRS\ (0 GHDOVZLWKWKHHOXFLGDWLRQRIĺQHVWUXFWXUDOIHDWXUHV XQGHUO\LQJELRORJLFDORUJDQL]DWLRQ7KHHOHFWURQPLFURVFRSHKDVPDGHRXWVWDQ ding contributions by illuminating the morphological details which are not clearly REVHUYHGE\WKHYHQHUDEOHOLJKWPLFURVFRSH7KXV(0KDVHQKDQFHGRXUXQGHUV WDQGLQJRIWKHEDVLFRUJDQL]DWLRQRIWKHFHOO)XUWKHUPRUH(0KDVEHHQRIJUHDW use in unravelling the organization of complex synaptic relations between neurons, in analysing the ultrastructure of pathogens and in examining the morphology of various cellular organelles such as mitochondria, rough endoplasmic reticulum DQG*ROJL +LVWRULFDOO\(0KDVORQJEHHQXVHGLQWKHFKDUDFWHUL]DWLRQRIUDELHVYLUXV 5$%9 LQWKHFHQWUDOQHUYRXVV\VWHP &16 1HJULERGLHVDQG5$%9SDUWLFOHVKDYHEHHQ IRXQGLQQHXURQVDQGQHXURQDOSURFHVVHVLQ&16WLVVXHVLQFOXGLQJ$PPRQpVKRUQ RIWKHKLSSRFDPSXVDQGWKHFHUHEHOOXPEUDLQVWHPDQGFHUHEUDOWLVVXHV8VLQJ WKLQVHFWLRQ(0LQ 0DWVXPRWR (1)FRQĺUPHGWKHYLUDOQDWXUHRIWKH1HJUL ERGLHV GHVFULELQJ D PDWUL[ FRQWDLQLQJ HORQJDWHG SDUWLFOHV 7KH PRUSKRORJ\ RI WKHYLUDOQXFOHRFDSVLGZDVIRXQGE\QHJDWLYHVWDLQ(0WREHDVLQJOHVWUDQGHG helix (2),PPXQRĻXRUHVFHQFHDQG(0ZHUHLQVWUXPHQWDOLQXQGHUVWDQGLQJWKH SDWKRJHQHVLVRI5$%9LQH[SHULPHQWDODQLPDOV(3)$OVR(0KDVDVVLVWHGLQWKH GHVFULSWLRQRIFDVHVLQZKLFK5$%9ZDVWUDQVPLWWHGE\FRUQHDOWUDQVSODQWDWLRQ and of solid organs from infected donors to recipients (4, 5)1HJULERGLHVZHUH VKRZQ WR FRQWDLQ 1 DQG 3 SURWHLQV E\ LPPXQRJROG (0 DQG YLUDO P51$ DQG JHQRPLFDQGDQWLJHQRPLF51$ZHUHGHWHFWHGE\ĻXRUHVFHQWLQVLWXK\EULGL]DWLRQ (6) (0KDVDOVREHHQXVHGWRFRQĺ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t7KLVFKDSWHUGHVFULEHVDSURWRFROIRUWKHXVHRI7(0LQ UHODWHGVWXGLHV Transmission electron microscopy Laboratory techniques in rabies Fifth edition Part 2. 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Detection of virus #NNGW$TėGRSĂWCěKUGSSěCKNSCNF embedding media Buffers 0.1 mol sodium phosphate buffer Recipe 1 6ROXWLRQ$ 'LVVROYHJ1D+POLQP/RITXDUW]GRXEOHGLVWLOOHGZDWHU 6ROXWLRQ% 'LVVROYHJ1D+POLQP/RITXDUW]GRXEOHGLVWLOOHGZDWHU 3UHSDUHPROSKRVSKDWHEXIIHUE\DGGLQJ UDWLRRIVROXWLRQ$DQG%UHVSHF WLYHO\$GMXVWWKHS+WRE\DGGLQJVROXWLRQ$RU% Recipe 2 'LVVROYH JVRGLXPSKRVSKDWH PRQREDVLF DQGJVRGLXPSKRVSKDWH GLEDVLF LQP/G+2$GMXVWWKHS+WRZLWKOLTXLGVRGLXPK\GUR[LGH 0.1 mol sodium cacodylate buffer 'LVVROYH J VRGLXP FDFRG\ODWH LQ P/ RI TXDUW] GRXEOH GLVWLOOHG ZDWHU $GGJRIVXFURVHDQGPL[XQWLOLWGLVVROYHV$GMXVWS+ZLWK1+&/ 7KLV}VROXWLRQLVVWDEOHIRUGD\VDW& )L[DWLYHV(these solutions are stable for 1 month at 4 °C) 3% glutaraldehyde: glutaraldehyde is often supplied as 25% s JOXWDUDOGHK\GH P/ s PROVRGLXPSKRVSKDWH RUPROVRGLXPFDFRG\ODWHEXIIHU P/ 2.5% glutaraldehyde 2WKHU GLVWULEXWRUV VXSSO\ JOXWDUDOGHK\GH DV LQ D P/ DPSRXOH VHDOHG XQGHUGU\QLWURJHQ s JOXWDUDOGHK\GH P/ s PROVRGLXPSKRVSKDWH RUPROVRGLXPFDFRG\ODWH P/ 5% paraformaldehyde s (0JUDGHSDUDIRUPDOGHK\GH P/ s PROVRGLXPSKRVSKDWH RUPROVRGLXPFDFRG\ODWH P/ Transmission electron microscopy Laboratory techniques in rabies Fifth edition Part 2. Detection of virus Stains 1% toluidine blue 'LVVROYH J VRGLXP WHWUDERUDWH %RUD[ RU VRGLXP FDUERQDWH LQ P/ KRW GLVWLOOHGZDWHU & EULQJGRZQWRURRPWHPSHUDWXUHWKHQDGGJWROXLGLQH EOXH6WLUZHOOXQWLOLWGLVVROYHVDQGĺOWHUWKHVROXWLRQEHIRUHXVHE\XVLQJDV\ULQJH ĺOWHU Uranyl acetate Several different uranyl acetate solutions have been employed for staining ultra WKLQ VHFWLRQV $OFRKROLF VROXWLRQV JLYH EHWWHU SHQHWUDWLRQ RI VWDLQ LQWR VHFWLRQV WKDQDTXHRXVVROXWLRQV3UHSDUHVDWXUDWHGXUDQ\ODFHWDWHVROXWLRQLQHWKDQRO DQGNHHSLQDEURZQERWWOHRUDZD\IURPOLJKW7DNHP/RIVROXWLRQDQGFHQWUL IXJHEHIRUHXVHRUXVHDV\ULQJHDQGV\ULQJHĺOWHU Lead citrate 'LVVROYHPJRIOHDGFLWUDWHLQ P/GLVWLOOHGZDWHU$GGtJVRGLXP K\GUR[LGHWRGLVVROYHOHDGFLWUDWHWKHQDGGGLVWLOOHGZDWHUWRPDNHXSP/ S+} Embedding media ,QJUHGLHQWVDUHDGGHGDQGPL[HGWKRURXJKO\5HPRYHDLUEXEEOHVE\KHDWLQJDW }&IRUPLQRUE\SODFLQJXQGHUDYDFXXP Preparation of Araldite CY 212 embedding medium s DUDOGLWH&<tHSR[\UHVLQ P/ s ''6$ GRGHFHQ\OVXFFLQLFDQK\GULGH tKDUGHQHU P/ s GLEXW\OSKWKDODWHtSODVWLFL]HU P/ s '03 WULVGLPHWK\ODPLQRPHWK\OSKHQRO tDFFHOHUDWRU P/ Preparation of Epon-substitute/Araldite 502 embedding media s (SRQVXEVWLWXWH P/ s DUDOGLWH P/ s ''6$ GRGHFHQ\OVXFFLQLFDQK\GULGH P/ s GLEXW\OSKWKDODWH P/ s '03 WULVGLPHWK\ODPLQRPHWK\OSKHQRO P/ RURI'03 Transmission electron microscopy Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens Part 3. Demonstration of antigens Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens Chapter 11 6JGFKRGEěĝTORGSEGNěCNěKDOFXěGSě Introduction Historically, before the advent of modern laboratory techniques, diagnosis of rabies was based upon clinical signs (1)'XULQJWKHODWHVQRQVSHFLĺF KLVWRORJLFDO HYLGHQFH RI FHQWUDO QHUYRXV V\VWHP &16 LQĻDPPDWLRQ DQG ODWHU GHPRQVWUDWLRQ RI LQWUDF\WRSODVPLF LQFOXVLRQV ZLWKLQ QHXURQV VXFK DV 1HJUL bodies formed a basis for early laboratory diagnosis, coupled with accompanying EHKDYLRXUDODOWHUDWLRQVRUDKLVWRU\RIDELWHIURPDVXVSHFWUDELGDQLPDO%DVHG upon the predilection of lyssaviruses for infection of the CNS, the brain became WKHRUJDQRIFKRLFHIRUURXWLQHGLDJQRVLVRIUDELHV*UDGXDOO\WKHFRPELQDWLRQRI clinical observations, improved laboratory techniques and epidemiological surveil lance resulted in the accumulated evidence that, without primary replication in the CNS, dissemination to other anatomical sites did not occur, including the salivary JODQGV$VWKHPDMRUSRUWDORIYLUDOH[LWWKHVDOLYDU\JODQGVPLJKWVHHPWRKDYH been a more preferable tissue than the CNS for laboratory diagnosis, except that multiple glands contribute to the composition of saliva and salivary excretion was NQRZQWREHLQWHUPLWWHQW Hence, salivary glands were not selected as appropriate samples for primary GLDJQRVWLFIRFXV%\WKHODWH V*ROGZDVVHUDQG.LVVOLQJKDGGHPRQVWUDWHG WKHXWLOLW\RIWKHĻXRUHVFHQWDQWLERG\WHFKQLTXHVLQGHWHFWLQJUDELHVYLUXV 5$%9 antigens in the CNS of rabid animals (2)7KHIXQGDPHQWDOSULQFLSOHRIWKHWHVW GHSHQGV XSRQ WKH PLFURVFRSLF GHWHFWLRQ RI YLUDO DQWLJHQV XVLQJ ĻXRUHVFHLQ LVRWKLRF\DQDWH ),7& FRQMXJDWHG DQWLERGLHV WR 5$%9,Q WKH HQVXLQJ GHFDGHV IXUWKHULPSURYHPHQWVWRWKHGLUHFWĻXRUHVFHQWDQWLERG\WHVW ')$7 ZHUHPDGH and greater experience was obtained in its application as a standard diagnostic technique (3, 4, 5)%\WKHVWFHQWXU\WKH')$7ZDVHPSOR\HGIRUGLDJQRVLVRI rabies in millions of cases of suspect animals and was documented to detect DOOO\VVDYLUXVHV$VERWKWKHVHQVLWLYLW\DQGWKHVSHFLĺFLW\RIWKH')$7DSSURDFK LWUHPDLQVWKHJROGVWDQGDUGIRUURXWLQHYHWHULQDU\DQGKXPDQODERUDWRU\ diagnosis of rabies today (6, 7) 7KLVFKDSWHUGHVFULEHVRQHVWDQGDUGRSHUDWLQJSURFHGXUHWKDWGHWDLOVSUHDQD O\WLFDO DQDO\WLFDO DQG SRVWDQDO\WLFDO IDFHWV RI WKH ')$7 EDVHG RQ WKH 8QLWHG States Protocol for postmortem diagnosis of rabies in animals by direct fluorescent anti- body testing (8) The brainstem, for example pons, medulla and thalamus, is the most critical WLVVXH IRU GLDJQRVWLF WHVWLQJ RI UDELHV 7KH 5$%9 DQWLJHQV DUH ZLGHVSUHDG WKURXJKRXWWKHEUDLQLQPRVWUDELGVXEMHFWV+RZHYHUWKRVHZLWKRQO\DVSDUVH amount of viral antigens tend to have more present in the brainstem than in the RWKHU&16UHJLRQV)XUWKHUPRUHH[DPLQDWLRQRIDFRPSOHWHFURVVVHFWLRQRIWKH brainstem tests the maximum ascending and descending nerve tracts, which is #HQDBSkTNQDRBDMS@MSHANCXSDRS Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens LPSRUWDQWEHFDXVHRIWKHSRVVLELOLW\RIXQLODWHUDOVSUHDGRI5$%9SDUWLFXODUO\LQ ODUJHDQLPDOV )LJ $ORQJZLWKWKHEUDLQVWHPHLWKHUFHUHEHOODURUKLSSR FDPSXVWLVVXHPD\EHH[DPLQHG$PRUHFRQĺGHQWGLDJQRVLVPD\EHDFKLHYHG E\ WHVWLQJ DGGLWLRQDO WLVVXH IURP RQH RU ERWK RI WKHVH UHJLRQV EXW D GHĺ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ě Proper collection, shipping and handling of rabies specimens is essential for D UDSLG DQG UHOLDEOH GLDJQRVLV RI UDELHV LQ DQLPDOV ,Q HDFK ORFDOLW\VXEPLWWHUV VKRXOGEHSURYLGHGZLWKJXLGHOLQHVIRUSURSHUSDFNDJLQJRIVXVSHFWVSHFLPHQV DQGVKLSSLQJUHTXLUHPHQWV,QWDFWKHDGVRUHQWLUHFDUFDVVHV LIDVPDOOPDPPDO VXFKDVDEDW PD\EHVXEPLWWHGWRWKHODERUDWRU\)UHVKXQĺ[HGGLVVHFWHGEUDLQ tissue is acceptable but must include a complete transverse cross section of the brainstem and aliquots of tissue from the cerebellum (vermis, right and left) lobes if DYDLODEOH,IWKHFHUHEHOOXPLVXQDYDLODEOHIRUWHVWLQJWKHQWKHULJKWDQGOHIWKLSSR FDPSLPD\EHVXEVWLWXWHG&DUHPXVWEHWDNHQWRDYRLGWUDXPDWRWKHEUDLQGXULQJ HXWKDQDVLD RU VDPSOH SURFHVVLQJ $ VXEPLVVLRQ IRUP VKRXOG DFFRPSDQ\ WKH VSHFLPHQDQGVKRXOGLQFOXGHEDVLFLQIRUPDWLRQVXFKDVWKHVXEPLWWHUpVQDPHDQG FRQWDFWLQIRUPDWLRQDQLPDOW\SHDQGLGHQWLĺHUGDWHRIFROOHFWLRQKLVWRU\RIH[SR #HQDBSkTNQDRBDMS@MSHANCXSDRS Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens sure in the human or pet, for example bite or scratch, animal owner, geographical ORFDWLRQ DQG YDFFLQDWLRQ KLVWRU\6DPSOH LGHQWLĺHUV DUH HVVHQWLDO ZKHQ VXEPLW ting more than one sample at a time, and specimens should be labelled clearly DQG SDFNDJHG VHSDUDWHO\ LQ OHDNSURRI FRQWDLQHUV 5DSLG GHOLYHU\ RU VKLSPHQW RIVSHFLPHQVWRWKHODERUDWRU\LVYLWDOHVSHFLDOO\LIKXPDQUDELHVSRVWH[SRVXUH SURSK\OD[LV 3(3 LVGHOD\HGSHQGLQJDODERUDWRU\UHVXOW6SHFLPHQVVKRXOGEH NHSWFKLOOHGQRWIUR]HQSULRUWRVKLSPHQWWRWKHSULPDU\WHVWLQJODERUDWRU\)UR]HQ heads must thaw before they can be safely necropsied, and freezing may delay WHVWLQJ7HVWLQJFDQRFFXURQIUR]HQWLVVXHVEXWUHSHDWHGIUHH]HtWKDZF\FOHVPD\ FRPSURPLVH WHVW UHVXOWV DQG VWRUDJH LQ IURVWIUHH IUHH]HUV VKRXOG WKHUHIRUH EH DYRLGHG Materials Reagents • acetone (of American Chemical Society grade) s FRQMXJDWH GLOXHQW VWHULOH PRO SKRVSKDWH EXIIHUHG VDOLQH S+ t ZLWKRSWLRQDO(YDQV%OXHFRXQWHUVWDLQt/P/ s ),7&FRQMXJDWHG DQWL5$%9 DQWLERG\ UHDJHQWV LQ WKH 86$ WZR GLIIHUHQW FRQMXJDWHVDUHUHTXLUHGLQHDFKWHVW s JO\FHUROt75,6EXIIHUFRYHUVOLSPRXQWLQJĻXLG VHH$QQH[ s SKRVSKDWHEXIIHUHGVDOLQH 3%6 PROS+tIRUZDVKLQJVOLGHV s VSHFLĺFLW\ FRQWURO ),7&FRQMXJDWHG QRQ5$%9 DQWLERGLHV VSHFLHV DQG LVRW\SHPDWFKIRURQHDQWL5$%9FRQMXJDWH Supplies and equipment For necropsy/brain removal and dissection • autoclave s DEVRUEHQWEORWWHUV ELEXORXVSDSHUQDWXUDOSDSHUWRZHOVGLVSRVDEOHZLSHVHWF • bone chisel (Councilman) s FORVHGIURQWJRZQRUFRYHUDOOV s FRQWDLQHUVIRUDFHWRQHĺ[DWLRQDQG3%6VRDNV HJ&RSOLQMDUVSRO\SURS\ OHQHSODFHVOLGHKROGHUVVWDLQLQJGLVKHV • disinfectant (refer to Chapter 3RQ%LRVDIHW\ s GLVSRVDEOHODERUDWRU\PDWWRZHOVRUQHZVSDSHUVWRFRYHUZRUNVXUIDFH • face shield • forceps (small and large) • hammer • incinerator • large plastic bags for disposal of animal heads and waste s PLFURVFRSHVOLGHV LGHDOO\SRO\WHWUDĻXRURHWK\OHQH 37)( >7HĻRQ@FRDWHGRU SUHULQJHGZLWKWZRtPPGLDPHWHUZHOOVIRUURXWLQHWHVWVDQGWKUHH PPZHOOVIRUFRQĺUPDWRU\WHVWVZLWKIURVWHGHQGV • microscope slide boxes #HQDBSkTNQDRBDMS@MSHANCXSDRS Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens • necropsy apron (optional) • Petri dishes, paper plates or suitable containers for dissection of brain tissues s SRZGHUIUHHJORYHV QLWULOHRUODWH[ • restraining device (or method of securing animal heads) • rubber gloves (heavy duty) s VDPSOHFRQWDLQHUV HJSODVWLFVFUHZFDSWXEHVRUMDUVRLQWPHQWWLQV • scalpels • tongue depressors for lifting and retracting the brain For DFA testing s FRYHUVOLSV PP[PP s ĻXRUHVFHQFH PLFURVFRSH LGHDOO\ HTXLSSHG ZLWK +%2 : RU PHWDO KDOLGHLOOXPLQDWRU FXUUHQW/('tOHVVVXLWDEOH 3ODQ$SRFKURPDWOHQV[ !}}1$ DQG3ODQ)OXRULWH[ !1$RURLO ,QYHUWHGPLFURVFRSHV although useful for cell culture techniques, are not recommended for routine ')$WHVWLQJ s IUHH]HUV QRQIURVWIUHHŗ&ĻDPPDEOHVWRUDJHDQGŗ& s KXPLGLĺHGVOLGHFKDPEHU WUD\ZLWKOLGVRU3HWULGLVKPD\EHVXEVWLWXWHG s LQFXEDWRU & s UHIULJHUDWRU & • slide trays (stainless steel or other nonporous autoclavable) s V\ULQJHĺOWHUVORZSURWHLQELQGLQJPPRUPPGLDPHWHUP IRU DPRXQWV!P/DPPVL]HĺOWHULVUHTXLUHG s V\ULQJHVP/RUtP/UHVSHFWLYHO\ Biological materials 8Qĺ[HG EUDLQ LPSUHVVLRQ SRVLWLYH DQG QHJDWLYH FRQWURO VOLGHV VKRXOG EH SUHSDUHGLQDGYDQFHDQGPD\EHVWRUHGIRUXSWRPRQWKDWŗ&RUDWŗ& RUEHORZIRUXSWRPRQWKV s 5$%9LQGLJHQRXVSRVLWLYH tDQWLJHQGLVWULEXWLRQIURPWZRUDELHVYLUXV YDULDQWVVHHQLQWKHUHJLRQ QHHGHGIRUFRQMXJDWHWLWUDWLRQDQGURXWLQHWHVWLQJ s QHJDWLYHEUDLQWLVVXHVIRUFRQWUROVOLGHV Methods Once specimens are received at the testing laboratory, they are given an acces VLRQQXPEHUDQGDOOSHUWLQHQWLQIRUPDWLRQLVUHFRUGHG$OOVDPSOHVDQGDVVRFLDWHG materials, such as submission forms, animal heads, carcasses, slides, Petri dishes or paper plates and containers with saved reference material, are labelled carefully ZLWKDFFHVVLRQQXPEHUV8VHRISULQWHGODEHOVPD\EHEHVW([WUHPHFDUHPXVW EHWDNHQWRSUHYHQWFURVVFRQWDPLQDWLRQEHWZHHQVDPSOHVWKURXJKRXWWKHWHVWLQJ SURFHGXUH(DFKVDPSOHPXVWEHSURFHVVHGRQDFOHDQZRUNVXUIDFHZLWKDFOHDQ #HQDBSkTNQDRBDMS@MSHANCXSDRS Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens VHSDUDWHVHWRILQVWUXPHQWV7KHODERUDWRU\SHUVRQQHOSHUIRUPLQJWKHQHFURSV\ VKRXOGZHDUDSSURSULDWHSHUVRQDOSURWHFWLYHHTXLSPHQW 33( $WDPLQLPXPWKLV LQFOXGHVDFORVHGIURQWODERUDWRU\FRDWJRZQRUFRYHUDOOVKHDY\UXEEHUJORYHV RUGRXEOHJORYHVVKRHFRYHUVRUUXEEHUERRWVVDIHW\JODVVHVDQGDIDFHVKLHOG $1 PDVNVKRXOGEHZRUQLIDELRORJLFDOVDIHW\FDELQHWLVQRWXVHGIRUQHFURSV\ (see Chapter 3RQ%LRVDIHW\IRUDGGLWLRQDOLQIRUPDWLRQ 1HFURSV\RQHVDPSOHDWD WLPHWRDYRLGFURVVFRQWDPLQDWLRQ 0RVWDQLPDOEUDLQVFDQEHUHPRYHGXVLQJDKDPPHUDQGFKLVHOPHWKRG%UDLQ WLVVXHV EUDLQVWHPDQGFHUHEHOOXP IURPODUJH OLYHVWRFN DQLPDOVPD\EHUHWULHYHG YLD WKH IRUHPDQ PDJQXP XVLQJ D ORQJKDQGOHG VFDOSHO DQG VSDWXOD RU VSRRQ Smaller mammals, such as bats, can be processed easily with a scalpel or scis VRUVDQGIRUFHSV Protocol )RU DYHUDJH PHGLXPVL]HG DQLPDOV SODFH WKH VSHFLPHQ LQ D UHVWUDLQLQJ GHYLFHWRDQFKRUWKHKHDG 0DNH D PLGOLQH LQFLVLRQ WKURXJK WKH VNLQ DQG PXVFOH XVLQJ D VFDOSHO RU ERQLQJ NQLIH WKHQ UHPRYH WKH VNLQ DQG PXVFOH IURP HDFK VLGH WR IXOO\ H[SRVHWKHVNXOOFDSWRWKHOHYHORIWKHHDUV 3RVLWLRQDFKLVHOEODGHEHKLQGWKHH\HVRFNHWVDQGFXWLQWRWKHERQHE\ VWULNLQJWKHFKLVHOZLWKDKDPPHU 0DNHWZRODWHUDOFXWVDERYHWKHHDUVDQGDĺQDOFXWDWWKHEDFNRIWKHVNXOO ZKHUHWKHVSLQDOFRUGHQWHUV8VHERQHFXWWHUVLIQHHGHGWRFXWWKURXJK DQ\DGKHULQJERQH%RQHFXWWHUVDUHHVVHQWLDOIRUFDWVSHFLPHQVVLQFHWKH FHUHEHOOXPLVHQFDVHGLQERQH Cut away dura mater, the membrane covering the brain, then sever the XQGHUO\LQJQHUYHVDQGFXWWKHEUDLQVWHPUHJLRQQHDUWKHVSLQDOFRUG Lift the brain out using forceps and a tongue depressor and place it in a VWHULOH3HWULGLVKRULQDQRWKHUVXLWDEOHFRQWDLQHU Notes: After necropsy of specimens, instruments must be handled appro priately; disposable scalpel blades and disposable plastic forceps must be discarded in a sharps container, and reusable stainless steel instruments GHFRQWDPLQDWHGE\SODFLQJLQGLVLQIHFWDQWDQGDXWRFODYHGEHWZHHQXVHV +HDGVRUFDUFDVVHVVKRXOGEHVDYHGXQWLOGLDJQRVWLFUHVXOWVDUHĺQDOL]HG DQGUHSRUWHGEHIRUHGLVFDUGLQJ If test results are unusual, it may be necessary to verify animal type or VSHFLHV$OOEUDLQWLVVXHVDPSOHVVKRXOGEHVDYHGIRUDWOHDVWPRQWKVDQG VWRUHGIUR]HQDWŗ&WRŗ&6RPHVSHFLPHQVVXFKDVWKRVHZLWK XQXVXDORUXQLTXHO\VVDYLUXVYDULDQWVVKRXOGEHVDYHGLQGHĺQLWHO\ Occasionally, samples demonstrating signs of decomposition are received IRUUDELHVWHVWLQJ$VDPSOHVKRXOGEHFRQVLGHUHGXQVDWLVIDFWRU\IRUWHVWLQJ LI L WKHFURVVVHFWLRQRIWKHEUDLQVWHPLVLQFRPSOHWH LL WKHEUDLQPDWHULDO is damaged beyond recognition; or (iii) the brain material is decomposed, for H[DPSOHOLTXHĺHGGLVFRORXUHG EURZQRUJUHHQ RUGHVLFFDWHG )LJ #HQDBSkTNQDRBDMS@MSHANCXSDRS Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens The brain material from unsatisfactory samples may be tested; however, LIQHJDWLYHWKHWHVWVKRXOGEHUHSRUWHGDVLQFRQFOXVLYHGXHWRLQVXIĺFLHQW VDPSOHRUVDPSOHFRQGLWLRQ VH&RQWURODQG3UHYHQWLRQDQG &KDUOHV(5XSSUHFKW/<66$//&$WODQWD*HRUJLD86$ %\FRXUWHV\RI/LOOLDQ2UFLDUL8QLWHG6WDWHV&HQWHUVIRU'LVHD )LJ'HFRPSRVHGFRZEUDLQ 6HYHUHDXWRO\VLVRIWLVVXHVLVQRWHGLQWKLVEUDLQVDPSOH7KHEUDLQWLVVXHVDUHGLVFRORXUHG GHPRQVWUDWLQJERWKEURZQ FHUHEHOOXP DQGJUHHQLVKFRORUDWLRQ FHUHEUDOKHPLVSKHUHV 7KH structural characteristics of the cerebellum are not recognizable; however, the structure of the FHUHEUXPLVVWLOOUHFRJQL]DEOH7KHEUDLQVWHP QRWVKRZQ ZDVOLTXLGXQUHFRJQL]DEOHDQGZDVQRW UHWULHYDEOH )ROORZLQJ QHFURSV\ GLVVHFW WKH DSSURSULDWH WLVVXHV QHHGHG IRU VOLGH SUHSDUDWLRQ ([WUDFWDFRPSOHWHFURVVVHFWLRQRIEUDLQVWHPDQGFHUHEHOOXP ULJKWOHIW DQGYHUPLV XVLQJDVWHULOHVFDOSHODQGIRUFHSV Place these pieces on an absorbent paper (bibulous paper, paper towel, HJ}.LPZLSH Prepare touch impressions by pressing a slide gently against the tissues on WKHSDSHUDQGEORWWLQJVHYHUDOWLPHVWRUHPRYHH[FHVVWLVVXH 0DNH GXSOLFDWH LPSUHVVLRQV IURP WKH VDPH SLHFH RI WLVVXH RQ WKH VDPH PLFURVFRSHVOLGH Note:$OZD\VEHFRQVLVWHQWZKHQPDNLQJLPSUHVVLRQVDQGWU\QRWWRORVH DQ\WLVVXH,IWKHEUDLQLVIURPDODUJHPDPPDOVXFKDVOLYHVWRFNLWPD\ UHTXLUHPXOWLSOHVOLGHVWRWHVWDIXOOFURVVVHFWLRQRIEUDLQVWHP #HQDBSkTNQDRBDMS@MSHANCXSDRS Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens &XWWKHFURVVVHFWLRQVLQKDOIWKHQPDNHGXSOLFDWHLPSUHVVLRQVIURPHDFK KDOI 3ODFHXQXVHGSRUWLRQVRIWKHEUDLQVWHPDQGFHUHEHOOXPLQDQRQEUHDNDEOH FRQWDLQHUVXFKDVDQRLQWPHQWWLQRUSODVWLFVFUHZFDSMDU 3ODFHWKHVOLGHVIURPHDFKVSHFLPHQLQDVHSDUDWHFRQWDLQHUIRUĺ[DWLRQDQG DOORZWRDLUGU\IRUDSSUR[LPDWHO\tPLQSULRUWRĺ[LQJ :KHQ VOLGHV DUH FRPSOHWHO\ GU\ DGG FKLOOHG ŗ & DFHWRQH WR HDFK FRQWDLQHUIRUĺ[DWLRQ )L[DQHJDWLYHFRQWUROVOLGHDQGDSRVLWLYHFRQWUROVOLGHDWWKHVDPHWLPHDV WKHWHVWVOLGHVDOVRLQVHSDUDWHFRQWDLQHUV3RVLWLYHFRQWUROVOLGHVVKRXOGEH made from tissue from a strongly positive specimen infected with the most FRPPRQ5$%9YDULDQWSUHVHQWLQWKHDUHD1HJDWLYHFRQWUROVOLGHVVKRXOG EHPDGHIURPDVSHFLPHQWKDWWHVWHGQHJDWLYHDQGKDGPLQLPXPEDFN JURXQGDQGQRQVSHFLĺFĻXRUHVFHQFH SRVLWLYHDQGQHJDWLYHFRQWUROVOLGHV can be made from the brain stem or cerebellum tissue, air dried and stored IUR]HQDWŗ&IRUXSWRPRQWKV )L[WHVWDQGFRQWUROVOLGHVLQDFHWRQHIRUDWOHDVWKWRRYHUQLJKWDWŗ& QRPRUHWKDQK )ROORZLQJĺ[DWLRQGLVFDUGDFHWRQHDSSURSULDWHO\LQWRDZDVWHERWWOHDQG DOORZVOLGHVWRGU\DWURRPWHPSHUDWXUH 3ODFH WKH VOLGHV LQ D PRLVWXUH FKDPEHU7ZRDQWL5$%9 UHDJHQWV SUHIH UDEO\WZRGLIIHUHQWPRQRFORQDOFRQMXJDWHVDUHXVHGIRU')$WHVWLQJ8VH RIWZRGLIIHUHQWFRQMXJDWHSURGXFWVUHGXFHVWKHFKDQFHRIQRQUHFRJQLWLRQ RIDQ\RQHYLUDOYDULDQW3RO\FORQDODQWLUDELHVUHDJHQWVDUHDOVRDQRSWLRQ EXW QRQVSHFLĺF ELQGLQJ FDQ EH SUREOHPDWLF EHFDXVH RI WKH SUHVHQFH RI H[WUDQHRXVDQWLERGLHV 7RSUHSDUHIUHVKZRUNLQJGLOXWLRQRIFRQMXJDWHUHPRYHWKHVWRFNFRQMXJDWH DOLTXRWVDQGPL[ZLWKWKHDSSURSULDWHDPRXQWRIGLOXHQW 3%6SOXVRSWLRQDO (YDQVEOXH WRREWDLQWKHZRUNLQJGLOXWLRQSUHYLRXVO\GHWHUPLQHGE\WLWUD WLRQ7KHZRUNLQJGLOXWLRQVRIFRQMXJDWHVKRXOGEHVWRUHGDW&DQGXVHG ZLWKLQGD\VRUGLVFDUGHG )LOO WZR V\ULQJHV ZLWK WKH ZRUNLQJ GLOXWLRQV RI WKH WZR DQWLUDELHV FRQMX JDWHVWKHQHTXLSHDFKV\ULQJHZLWKDPORZSURWHLQELQGLQJĺOWHU 'LVFDUGWKUHHGURSVRIFRQMXJDWHWKURXJKWKHĺOWHURIWKHV\ULQJHFRQWDLQLQJ WKHĺUVWDQWL5$%9UHDJHQWWKHQDGPLQLVWHUE\ĺOWHULQJRQWRWKHLPSUHVVLRQ LQWKHĺUVWZHOO FORVHVWWRWKHIURVWHGHQG RIDOOWKHVOLGHV 6WDUW DGGLQJ FRQMXJDWH WR WKH SRVLWLYH FRQWURO VOLGH IROORZ ZLWK WKH WHVW VOLGHVDQGODVWDGGWRWKHQHJDWLYHFRQWUROVOLGH0DNHVXUHWKHUHDJHQW FRPSOHWHO\FRYHUVWKHWRXFKLPSUHVVLRQV LWPD\WDNHtGURSVGHSHQGLQJ RQWKHVL]HRIHDFKZHOO $GGWKHVHFRQGDQWL5$%9UHDJHQWWRWKHGXSOLFDWHLPSUHVVLRQLQWKHRWKHU ZHOO IXUWKHVWIURPWKHIURVWHGHQGRIVOLGHV LQWKHVDPHPDQQHU #HQDBSkTNQDRBDMS@MSHANCXSDRS Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens ,QFXEDWHWKHVOLGHVLQDKXPLGLĺHGFKDPEHUDW&IRUPLQ )LJ Notes:,WLVHVVHQWLDOWKDWVXIĺFLHQWFRQMXJDWHLVFRYHULQJWKHVOLGHVDQGWKDW consistent humidity is maintained while slides are incubating so that the FRQMXJDWHGRHVQRWGU\RQWKHVOLGHV'ULHGFRQMXJDWHPD\EHPLVWDNHQIRU VSHFLĺFVWDLQLQJRUIRUREVFXUHVSHFLĺFVWDLQLQJRIO\VVDYLUXVDQWLJHQV $WODQWD*HRUJLD86$ VH&RQWURODQG3UHYHQWLRQDQG&KDUOHV(5XSSUHFKW/<66$//& %\FRXUWHV\RI/LOOLDQ2UFLDUL8QLWHG6WDWHV&HQWHUVIRU'LVHD )LJ2YHUYLHZRIWKHVWDQGDUGGLUHFWĻXRUHVFHQWDQWLERG\WHVW ')$7 7KHEDVLFVWHSVLQWKH')$7DUHUHYLHZHGLQWKLVĺJXUH,ISUHVHQW5$%9DQWLJHQV HJ513 LQDFHWRQHĺ[HGEUDLQ LPSUHVVLRQVZLOOUHDFWZLWKWKHWZR),7&ODEHOOHGDQWLUDELHVFRQMXJDWHVDQGIRUPVSHFLĺFDQWLJHQtDQWLERG\FRPSOH[HV $IWHUXQUHDFWHGDQWLUDELHVDQWLERGLHVDUHUHPRYHGE\ZDVKLQJZLWK3%6VSHFLĺFĻXRUHVFHQFH5$%9LQFOXVLRQVDUH REVHUYHG,IQR5$%9DQWLJHQVDUHSUHVHQWWKHQQRDQWLJHQtDQWLERG\FRPSOH[HVDUHIRUPHGDQGQRVSHFLĺFĻXRUHVFHQFH LHQRLQFOXVLRQV DUHREVHUYHG #HQDBSkTNQDRBDMS@MSHANCXSDRS Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens 5HPRYHVOLGHVIURPWKHLQFXEDWRUDQGJHQWO\ULQVHHDFKZHOORIWKHVOLGH FDUHIXOO\ZLWKDVWUHDPRI3%6IURPDZDVKERWWOH'RQRWDOORZFRQMXJDWH IURPRQHZHOOWRZDVKLQWRDQRWKHU Transfer each set of slides from a specimen to a rinse container; use one ULQVH FRQWDLQHU SHU VSHFLPHQ 8VH VHSDUDWH FRQWDLQHUV IRU WKH SRVLWLYH FRQWURODQGWKHQHJDWLYHFRQWURO 6RDNWKHVOLGHVLQ3%6IRUPLQ 'LVFDUGDQGUHSODFHZLWKIUHVK3%6DQGVRDNIRUDQRWKHUPLQ 5HPRYH VOLGHV IURP WKH 3%6 RQH E\ RQH DQG LQYHUW WKHP RQ WR FOHDQ DEVRUEHQWSDSHUWXUQWKHPRYHUWREORWWKHEDFNRIWKHVOLGHVDQGSODFH WKHPLQDVOLGHWUD\ $GG ORZ JO\FHURO 75,6EXIIHUHG PRXQWLQJ ĻXLG WR HDFK RI WKH FRYHUVOLSVĺUVWQRWGLUHFWO\WRWKHVOLGHXVLQJP/V\ULQJHĺWWHGZLWKD PPP 1DOJHQH V\ULQJHĺOWHU ,QYHUWWKHFRYHUVOLSZLWKPRXQWLQJPHGLXPRQWRWKHVOLGH7KHPRXQWLQJ PHGLXPXVHGLQWKH')$7PD\DIIHFWWKHDYLGLW\RIWKHDQWLJHQDQWLERG\ complex; mounting solutions with high glycerol concentrations are not recommended (9) Note: ,I WKH DERYHPHQWLRQHG PRXQWLQJ PHGLXP LV QRW XVHG WR DWWDFK coverslips to slides, small inclusions and sparse amounts of antigen may EHPLVVHG )LJ 7DNHWKHVOLGHVLQWRWKHGDUNURRPHTXLSSHGZLWKDĻXRUHVFHQFHPLFURV FRSH .HHSWKHVOLGHVLQWKHGDUNDQGREVHUYHWKHWHVWVOLGHVZLWKLQKXVLQJD ĻXRUHVFHQFHPLFURVFRSH,IVOLGHVZLOOQRWEHUHDGZLWKLQKWKH\FDQEH VWRUHGDWŗ&RUEHORZDQGUHWDLQĻXRUHVFHQFH ([DPLQH LPSUHVVLRQV RQ VOLGHV WKRURXJKO\ DW [ 3ODQ$SRFKURPDW ł} } 1$ PDJQLĺFDWLRQ IRU VSDUNOLQJ DSSOHJUHHQ ĻXRUHVFLQJ LQFOX VLRQV If further resolution is required to examine the inclusion morphology, REVHUYHZLWK[QHRĻXRULWHOHQVZLWKł1$,GHDOO\WZRUHDGHUVVKRXOG examine the slides made from each test animal to ensure correct interpreta WLRQ5HVXOWVDUHUHFRUGHGIRUWKHFRQWUROVOLGHVDQGIRUHDFKLPSUHVVLRQRI WKHWHVWVOLGHVRQDZRUNVKHHW7KHUHDFWLRQVDUHUDWHGRQVWDLQLQJLQWHQVLW\ DQGDQWLJHQGLVWULEXWLRQ)RUH[DPSOH5$%9DQWLJHQVWKDWVWDLQVSDUNOLQJ DSSOHJUHHQĻXRUHVFHQFHDUHUDWHGDVLQWHQVLW\6XEVWDQGDUGVDPSOHV such as those with some degree of decomposition, might have a slightly GLPLQLVKHGEULJKWQHVVEULJKWEXWDUHQRWVSDUNOLQJ'XOOJUHHQLQFOX VLRQVZLWKRUOHVVDUHQRWGLDJQRVWLFRIUDELHVXQOHVVDGGLWLRQDOWHVWLQJ FRQĺUPVWKHLQFOXVLRQVDUHO\VVDYLUXVVSHFLĺF #HQDBSkTNQDRBDMS@MSHANCXSDRS Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens FDI conjugate Coverslip with low glycerol TRIS buffered Sigma Millipore 5100 mounting medium Coverslip with low glycerol TRIS buffered No coverslip and mounting medium mounting medium %\FRXUWHV\RI/LOOLDQ2UFLDUL8QLWHG6WDWHV&HQWHUVIRU'LVHDVH&RQWURODQG3UHYHQWLRQDQG&KDUOHV(5XSSUHFKW/<66$//&$WODQWD*HRUJLD86$ )LJ')$7SRVLWLYHLPSUHVVLRQVZLWKDQGZLWKRXWORZJO\FHUROPRXQWLQJPHGLXP 'XSOLFDWHEUDLQLPSUHVVLRQVIURPWKHVDPHSRVLWLYH 6RXWK&HQWUDO6NXQN5$%9 VDPSOHZHUHWHVWHGZLWKZRUNLQJGLOXWLRQVRIDQWL UDELHVFRQMXJDWHV )XMLUHELR'LDJQRVWLFV,QFDQG6LJPD0LOOLSRUH')$ 3KRWRJUDSKVZHUHWDNHQRIEUDLQLPSUHVVLRQVOLGHVZLWK DQGZLWKRXWFRYHUVOLSVDQGPRXQWLQJPHGLXPDW[PDJQLĺFDWLRQ Positive control Negative control %\FRXUWHV\RI/LOOLDQ2UFLDUL8QLWHG6WDWHV&HQWHUV IRU'LVHDVH&RQWURODQG3UHYHQWLRQDQG&KDUOHV( 5XSSUHFKW/<66$//&$WODQWD*HRUJLD86$ )LJ')$7FRQWUROV 7KHSRVLWLYHFRQWUROLPSUHVVLRQ OHIW GHPRQVWUDWHVVSDUNOLQJDSSOHJUHHQĻXRUHVFHQFHDQGtDQWLJHQGLVWULEXWLRQ7KHZRUNLQJ GLOXWLRQRIFRQMXJDWHVKRXOGEHGLOXWHGVRWKDWLQWHQVHDSSOHJUHHQĻXRUHVFHQFHLVREVHUYHGLQWKHSRVLWLYHFRQWUROIRUHDFKWHVW LQFOXGLQJREVHUYDWLRQRIRIWKHVPDOODQGODUJHLQFOXVLRQV7KHQHJDWLYHFRQWUROVOLGHGHPRQVWUDWHVQRVSHFLĺFĻXRUHVFHQFH #HQDBSkTNQDRBDMS@MSHANCXSDRS Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens 7KHGLVWULEXWLRQRIDQWLJHQVLVJUDGHGRQDVFDOHRIGRZQWR,IUDWHG DV}LQFOXVLRQVRIYDU\LQJVL]HDQGVKDSHDUHREVHUYHGLQQHDUO\DOOPLFURVFRSH ĺHOGVDQGLQFOXVLRQVRIYDU\LQJVL]HDQGVKDSHDUHQXPHURXVLQPRVWĺHOGV +RZHYHULIUDWHGDVtRIWKHĺHOGVKDYHLQFOXVLRQVRIYDU\LQJVL]HDQG VKDSHDQGKDYHLQFOXVLRQVRIYDU\LQJVL]HDQGVKDSHVHHQLQ RIWKH PLFURVFRSHĺHOGV 5HDGHUVVKRXOGUHFRUGWKHDQWLJHQLQWHQVLW\GLVWULEXWLRQIRUHDFKLPSUHVVLRQ IRUH[DPSOHVRUDQGPDNHDQRWHLIWKHVWDLQLQJLQWHQVLW\LV EHORZ 7KHSRVLWLYHFRQWUROVOLGHVPXVWGHPRQVWUDWHWKHH[SHFWHGVWDLQLQJLQWHQ VLW\tDQWLJHQGLVWULEXWLRQ )LJ 7KHUHVKRXOGEHQRVWDLQLQJRQWKHQHJD WLYHFRQWUROVOLGHV7KHWHVWVOLGHVPXVWEHUHPDGHDQGUHWHVWHGLIWKHSRVLWLYHDQG QHJDWLYHFRQWUROVOLGHVGRQRWGHPRQVWUDWHDFFHSWDEOHUHVXOWV Interpretation of test results Test results may be reported as negative for rabies, positive for rabies, unsa WLVIDFWRU\RULQFRQFOXVLYH,IERWKVOLGHVDUHFOHDUO\QHJDWLYHIRUWKHSUHVHQFHRI lyssavirus antigens, the sample is reported out as negative for rabies, provided WKH SURSHU WLVVXHV ZHUH WHVWHG DQG LQ VDWLVIDFWRU\ FRQGLWLRQ ,I ERWK VOLGHV DUH FOHDUO\SRVLWLYHKDYLQJDWOHDVWDWRLQWHQVLW\DQGWRDQWLJHQGLVWULEX WLRQWKHVDPSOHLVUHSRUWHGRXWDVSRVLWLYHIRUUDELHV6RPHWLPHVLQH[SHULHQFHG LQGLYLGXDOVPD\EHFRQFHUQHGZKHQREVHUYLQJ\HOORZRUJROGHQDXWRĻXRUHVFHQFH LQQHJDWLYHVDPSOHVFDXVHGE\DFFXPXODWLRQVRIOLSRIXVFLQ )LJ 6SHFLPHQV WKDW DUH QRW FOHDUO\ QHJDWLYH RU SRVLWLYH UHTXLUH UHSHDW WHVWLQJ )RU}H[DPSOHVDPSOHVZLWKYHU\VSDUVHLQFOXVLRQVOHVVWKDQRIĺHOGVGHPRQV trating antigen, inclusions with atypical morphology or inclusions with staining LQWHQVLW\RIOHVVWKDQEULJKWQHVVZDUUDQWUHSHDWWHVWLQJ,IDIWHUUHSHDWWHVWLQJ ZLWKVSHFLĺFLW\FRQWUROFRQMXJDWHVWKHUHVXOWVDUHVWLOOQRWFOHDUWKHVSHFLPHQWHVW UHVXOW LV UHSRUWHG RXW DV LQFRQFOXVLYH $OWHUQDWLYH FRQĺUPDWRU\ WHVWLQJ PD\ EH QHFHVVDU\WRUXOHRXWUDELHV %HFDXVH3(3RIKXPDQUDELHVLVRIWHQGHOD\HGXQWLODWHVWUHVXOWLVDWWDLQHG UHSHDWWHVWLQJVKRXOGEHDFFRPSOLVKHGDVTXLFNO\DVSRVVLEOH5HSHDWVOLGHVDUH made from the original brain tissue that is saved after dissecting the brain and PDNLQJ WKH LQLWLDO VHW RI VOLGHV ,PSUHVVLRQV DUH PDGH IURP D FURVVVHFWLRQ RI EUDLQVWHP DQG FHUHEHOOXP +RZHYHU IRU WKH UHSHDW FRQĺUPDWRU\ ')$7 WKUHH LGHQWLFDOEUDLQLPSUHVVLRQVDUHPDGHIURPHDFKSLHFHRIWLVVXHSHUZHOOVOLGH 0XOWLSOHLPSUHVVLRQVOLGHVPD\EHUHTXLUHGWRWHVWHDFKRIWKHUHTXLUHGWLVVXHVIXOO FURVVVHFWLRQRIEUDLQVWHPDQGWKUHHOREHVRIFHUHEHOOXP ,QWKHUHSHDWFRQĺUPDWRU\')$7WKHEUDLQLPSUHVVLRQVOLGHVDUHWHVWHGZLWK WZRDQWLUDELHVFRQMXJDWHVDQGWKHVSHFLĺFLW\FRQWUROUHDJHQWQHJDWLYHFRQWURO FRQMXJDWH7KHVSHFLĺFLW\FRQWUROUHDJHQWFRQWDLQVQRQUDELHVDQWLERGLHVRIWKH VDPHLVRW\SHVSUHSDUHGLQWKHVDPHDQLPDODVWKHDQWLUDELHVFRQMXJDWHZKLFKLW LVSDLUHG #HQDBSkTNQDRBDMS@MSHANCXSDRS Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens %\FRXUWHV\RI/LOOLDQ2UFLDUL8QLWHG6WDWHV&HQWHUVIRU'LVHDVH&RQWURODQG3UHYHQWLRQDQG&KDUOHV(5XSSUHFKW/<66$//&$WODQWD*HRUJLD86$ )LJ5$%9SRVLWLYHDQWLJHQVFRPSDUHGZLWKDW\SLFDODXWRĻXRUHVFHQFH 7KHSRVLWLYHFRQWURO OHIW ZLWKVSDUNOLQJDSSOHJUHHQĻXRUHVFHQFHDQGGLVWULEXWLRQLVFRPSDUHGZLWKDW\SLFDODXWRĻXRUHVFHQFH REVHUYHGLQQHJDWLYHVDPSOH ULJKW ZLWKDFFXPXODWLRQVRIOLSRIXVFLQLQWKH&16WLVVXHLPSUHVVLRQ1RWHWKHJROGt\HOORZFRORXUDWLRQ RIREMHFWVZLWKRXWGLVWLQFWRXWOLQHVVHHQRFFDVLRQDOO\LQROGHUDQLPDOVDQGLQWKHFHUHEHOOXPRIFDWV7KLVJROGDXWRĻXRUHVFHQFHLV QRWLPPXQRĻXRUHVFHQFHRUQRQVSHFLĺFELQGLQJ7KHVDPHREVHUYDWLRQLVSRVVLEOHZLWKRXWVWDLQLQJZLWK),7&ODEHOOHGFRQMXJDWHVE\ H[SRVLQJWLVVXHLPSUHVVLRQVZLWKOLSRIXVFLQWREOXHOLJKW 1RWH )RU H[DPSOH FXUUHQWO\ LQ WKH 86$ 6LJPD 0LOOLSRUH /LJKW 'LDJQRVWLFV 5DELHV')$&DWFRQWDLQVDPL[WXUHRIWZRPRXVH,J*PRQRFORQDODQWL ERGLHVDQGRQHPRXVH,J*PRQRFORQDODQWLERG\),7&ODEHOOHG7KHVSHFLĺFLW\ FRQWURO UHDJHQW SDLUHG ZLWK WKLV DQWLUDELHV FRQMXJDWH LV 6LJPD 0LOOLSRUH /LJKW 'LDJQRVWLFV5DELHV1HJDWLYH&RQWURO 0RQRFORQDODQWLERG\),7&FRQMXJDWH &DWD ORJXH ,WLVDPL[WXUHRIWKHVDPHLVRW\SH,J*DQG,J*PRXVHPRQRFORQDO DQWLERGLHVDWWKHVDPHFRQFHQWUDWLRQLQPJP/DV6LJPD0LOOLSRUH&DW 3RVLWLYHDQGQHJDWLYHFRQWURO ZHOOVOLGHV PXVWEHWHVWHGLQFRQMXQFWLRQZLWK WKHUHSHDWWHVWVOLGHVXVLQJWZRDQWLUDELHVFRQMXJDWHVDQGWKHVSHFLĺFLW\FRQWURO UHDJHQWPHDQLQJWKHQHJDWLYHFRQWUROFRQMXJDWH )LJVt 7KHVSHFLPHQ is considered negative for rabies if all brain impressions are negative upon repeat WHVWLQJ ZLWK WZR DQWLUDELHV FRQMXJDWHV DQG WKH VSHFLĺFLW\ FRQWURO FRQMXJDWH )LJ} $OVRLIDW\SLFDOLQFOXVLRQVDUHSUHVHQWRQWHVWVOLGHVVWDLQHGZLWKRQH DQWLUDELHVFRQMXJDWHDQGWKHVSHFLĺFLW\FRQWUROSDLUHGZLWKWKDWFRQMXJDWHDQG QRLQFOXVLRQLVREVHUYHGZLWKWKHVHFRQGDQWLUDELHVFRQMXJDWHLQGLFDWLQJWKHVWDL QLQJLVQRQVSHFLĺF )LJ WKHWHVWLVFRQVLGHUHGQHJDWLYH The specimen is positive for lyssavirus antigens if, upon repeat testing, inclu sions typical of rabies are observed on impressions tested with one or both of the DQWL5$%9FRQMXJDWHVEXWQRWWKHVSHFLĺFLW\FRQWUROUHDJHQWSDLUHGWRRQHRIWKH DQWLUDELHVFRQMXJDWHV )LJV The specimen results are inconclusive if inclusions are observed on all impres VLRQVWHVWHGZLWKWKHWZRDQWLUDELHVFRQMXJDWHVDQGVSHFLĺFLW\FRQWUROVDQGDUH LQGLVWLQJXLVKDEOHIURP5$%9LQFOXVLRQVRUPD\PDVNVSHFLĺFVWDLQLQJ )LJ #HQDBSkTNQDRBDMS@MSHANCXSDRS Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens )LJ,IW\SLFDO5$%9DQWLJHQVĻXRUHVFHZLWK ERWKDQWLUDELHVFRQMXJDWHVDQGWKHVSHFLĺFLW\ control is negative, then the test is positive )LJ,IQRĻXRUHVFHQFHLVGHPRQVWUDWHG LQ')$ZLWKERWKDQWLUDELHVFRQMXJDWHVDQGWKH VSHFLĺFLW\FRQWUROWKHQWKH')$7LVQHJDWLYH )LJ,IFRQMXJDWHGHPRQVWUDWHVQR $WODQWD*HRUJLD86$ ĻXRUHVFHQFHDQGDW\SLFDOĻXRUHVFHQFHLV GHPRQVWUDWHGZLWKFRQMXJDWHDQGWKHVDPH ĻXRUHVFHQFHLVGHPRQVWUDWHGLQWKHVSHFLĺFLW\ FRQWUROSDLUHGZLWKFRQMXJDWHWKHQWKHWHVWFDQEH reported as negative )LJ,IDQWLUDELHVFRQMXJDWHGHPRQVWUDWHV QRĻXRUHVFHQFHEXWW\SLFDO5$%9DQWLJHQVĻXRUHVFH ZLWKFRQMXJDWHDQGWKHVSHFLĺFLW\FRQWUROLV negative, then the samples should be considered as positive, but sent to a reference laboratory for FRQĺUPDWLRQ VH&RQWURODQG3UHYHQWLRQDQG&KDUOHV(5XSSUHFKW/<66$//& )LJ,IDOOWKUHHFRQMXJDWHVGHPRQVWUDWH ĻXRUHVFHQFHLQGLVWLQJXLVKDEOHIURP5$%9DQWLJHQV then the test should be considered as indeterminate DQGDOWHUQDWLYHWHVWLQJLVUHTXLUHGWRUXOHRXW rabies; it may be necessary to send to a reference ODERUDWRU\IRUFRQĺUPDWLRQ %\FRXUWHV\RI/LOOLDQ2UFLDUL8QLWHG6WDWHV&HQWHUVIRU'LVHD )LJ&RQĺUPDWRU\')$7ZLWKVSHFLĺFLW\FRQWUROUHDJHQW #HQDBSkTNQDRBDMS@MSHANCXSDRS Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens $OWHUQDWLYH WHVWV VXFK DV UHDOWLPH 573&5'5,7 KHPLQHVWHG 573&5RU LVRODWLRQLQFHOOFXOWXUHPD\EHUHTXLUHGWRUXOHRXWUDELHV2IWKHDOWHUQDWLYHWHVWV UHDOWLPH573&5DQG'5,7DUHWKHPRVWXVHIXOIRUH[SHGLHQF\(10–12) Discussion 7KH')$7KDVSURYHQLWVXWLOLW\DVDQLGHDOGLDJQRVWLFPHWKRGGXULQJWKHSDVW \HDUVLQERWKGHYHORSHGDQGGHYHORSLQJFRXQWULHV(13)7KHWHVWLVKLJKO\VHQVL WLYHDQGVSHFLĺFDVGHPRQVWUDWHGLQPXOWLSOHH[WHUQDOLQWHUODERUDWRU\WULDOV(14) Owing to its accuracy and advantages in an anatomical–pathological context, in VRPHFRXQWULHVVXFKDVWKH86$QHDUO\VXVSHFWDQLPDOVPD\EHWHVWHG annually (15)3URSHUO\FRQWUROOHGWKHWHVWFDQSURYLGHUDSLGXQHTXLYRFDOUHVXOWV within a few hours, irrespective of lyssavirus species or mammalian taxa (including humans in either antemortem or postmortem situations) (7)7KHSURFHGXUHPD\ EHXVHGIRUDYDULHW\RISXUSRVHVLQFOXGLQJURXWLQHODERUDWRU\EDVHGVXUYHLOODQFH ULVNDVVHVVPHQWLQELWHH[SRVXUHFDVHVGHWHUPLQDWLRQRI5$%9LQH[SHULPHQWDO DQLPDOFOLQLFDOWULDOVLQYLWURDVVD\V VXFKDVWKHUDSLGĻXRUHVFHQWIRFXVLQKLELWLRQ WHVW>5)),7 VHHChapter 19@DQGWKHĻXRUHVFHQWDQWLERG\YLUXVQHXWUDOL]DWLRQWHVW >)$91 VHHChapter 20@DQGHYDOXDWLRQRIYDFFLQDWLRQSURJUDPPHV 8QOLNHWKH KLVWRULFDOXVHRIFRQYHQWLRQDO1HJULERG\LGHQWLĺFDWLRQXVLQJ6HOOHUVpVWDLQDQG the traditional process of holding suspect animals until late in the clinical course, UDELGDQLPDOVPD\EHGHWHFWHGXSRQWKHLQLWLDORQVHWRILOOQHVV)UHVKIUR]HQDQG ĺ[HGWLVVXHVFDQEHXVHG(16)&RQFHQWUDWLRQXSRQWKHEUDLQVWHPDQGFHUHEHOOXP as the CNS tissues of choice are based in part upon basic pathobiology, relative ease of anatomical access and economy of effort (17)%RWKPRQRFORQDODQGSRO\ FORQDOFRPPHUFLDO),7&FRQMXJDWHVFDQEHXWLOL]HG&XUUHQWO\LQWKH86$RQO\ PRQRFORQDODQWLERG\FRQMXJDWHVDUHDYDLODEOH 7DEOH ,QFDVHVRILQGHWHUPL QDWHUHVXOWVFRQĺUPDWRU\ODERUDWRU\WHVWLQJPD\LQYROYHYLUDOLVRODWLRQDOWHUQDWLYH antigen capture assays or relevant molecular tests (18) 3RWHQWLDOGUDZEDFNVLQFRQVLGHUDWLRQRIWKH')$7LQFOXGHWKHQHHGIRULQYHVW PHQW LQ D ĻXRUHVFHQFH PLFURVFRSH WKH QHFHVVLW\ IRU DSSURSULDWH KLJKTXDOLW\ KLJKLQWHQVLW\DUFODPSVUHJXODUPLFURVFRSHPDLQWHQDQFHDQGURXWLQHSDUWLFLSD WLRQLQSURĺFLHQF\WHVWLQJDVZHOODVWKHQHHGIRUXVHRILQIHFWLRXVDJHQWV 7DEOH0RQRFORQDODQWLERG\),7&ODEHOOHGFRQMXJDWHVIRUWKHUDELHV')$7LQWHUQDWLRQDOO\DYDLODEOH 0DQXIDFWXUHU Product name Product number Antibodies )XMLUHELR'LDJQRVWLFV )', ),7&$QWL5DELHV0RQRFORQDO &RQWDLQVDPL[WXUHRIWZR,J*D Globulin monoclonal antibodies 6LJPD0LOOLSRUH Light Diagnostics 705DELHV Contains the same mixture of two ')$5HDJHQW,, 0RQRFORQDO ,J*DPRQRFORQDODQWLERGLHVDV)', DQWLERG\),7&FRQMXJDWH 6LJPD0LOOLSRUH Light Diagnostics 705DELHV Contains the same mixture of two ')$5HDJHQW,,, 0RQRFORQDO ,J*DPRQRFORQDODQWLERGLHVDV)', DQWLERG\),7&FRQMXJDWH RQHDQWLERG\)DEIUDJPHQW 6LJPD0LOOLSRUH Light Diagnostics 705DELHV Contains the same mixture of two ')$5HDJHQW 0RQRFORQDO ,J*PRQRFORQDODQWLERGLHVDQGRQH DQWLERG\),7&FRQMXJDWH ,J*PRQRFORQDODQWLERG\ 6LJPD0LOOLSRUH Light Diagnostics 705DELHV &RQWDLQVDPL[WXUHRI,J*,J* 1HJDWLYH&RQWURO 0RQRFORQDO QRQUDELHVPRQRFORQDODQWLERGLHV DQWLERG\),7&FRQMXJDWH #HQDBSkTNQDRBDMS@MSHANCXSDRS Laboratory techniques in rabies Fifth edition Part 3. 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Demonstration of antigens Chapter 12 The direct rapid immunohistochemistry test for the detection of lyssavirus antigens Introduction 7KH GLUHFW UDSLG LPPXQRKLVWRFKHPLVWU\ WHVW '5,7 LV D VWUHSWDYLGLQtELRWLQ peroxidase staining technique for diagnosis of rabies by detecting lyssavirus DQWLJHQV 'XULQJ WKH SDVW GHFDGH WKH '5,7 KDV EHHQ XVHG H[WHQVLYHO\ IRU HQKDQFHGODERUDWRU\EDVHGVXUYHLOODQFHRIUDELHVLQ$IULFD$VLDDQGWKURXJKRXW WKH $PHULFDV ZLWK D VHQVLWLYLW\ DQG VSHFLĺFLW\ HTXLYDOHQW WR WKDW RI WKH GLUHFW ĻXRUHVFHQWDQWLERG\WHVW ')$7 DVGHVFULEHGLQChapter 11 and elsewhere (1–13) All relevant biosafety precautions should be followed as discussed in Chapter 3 RIWKLVPDQXDO Materials Equipment /LJKWPLFURVFRSHZLWKD[RFXODUDQGKLJKTXDOLW\REMHFWLYHOHQV HJ[[ Supplies • glass microscope slides s &RSOLQMDUVRUVWDLQLQJWUD\V • paper towels s qKXPLGLW\ FKDPEHUr D VLPSOH SODVWLF FRYHU SODFHG RYHU VOLGHV GXULQJ benchtop staining) • timer s PDUNLQJSHQVRUSHQFLOV s SLSHWWHV LQFOXGLQJ JODVV >RU FOHDU ORZWKHUPDOH[SDQVLRQ ERURVLOLFDWH 3\UH[@ • glass vials • centrifuge tube (or similar) • syringes s V\ULQJHĺOWHUV • pipette tips • pipetting device • microscope slide coverslips • positive and negative control tissue • scalpels Direct rapid immunohistochemistry test Laboratory techniques in rabies Fifth edition Part 3. 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Demonstration of antigens 9LHZVOLGHVE\OLJKWPLFURVFRS\XVLQJD[RFXODUDQG[REMHFWLYHOHQV WRVFDQWKHĺHOGWKRURXJKO\DQGD[ RUKLJKHU REMHFWLYHIRULQVSHFWLRQ DVQHHGHG1HJDWLYH&16VDPSOHVVWDLQEOXH )LJ ,QSRVLWLYH&16 VDPSOHV O\VVDYLUXV DQWLJHQV DSSHDU DV UHGGLVK LQWUDF\WRSODVPLF LQFOX VLRQV DJDLQVW D EOXH QHXURQDO EDFNJURXQG XVLQJ $(& DQG KDHPDWR[\OLQ FRXQWHUVWDLQ )LJVt )LJ([DPSOHRID/\VVDYLUXVQHJDWLYH&16VDPSOHVWDLQHGXVLQJWKH'5,7 a[PDJQLĺFDWLRQ %\FRXUWHV\RI&KDUOHV(5XSSUHFKW/<66$//6& USA Atlanta, Georgia, )LJ([DPSOHRID/\VVDYLUXVSRVLWLYH&16VDPSOHVWDLQHGXVLQJWKH'5,7 a[PDJQLĺFDWLRQ %\FRXUWHV\RI&KDUOHV(5XSSUHFKW/<66$//6& USA Atlanta, Georgia, )LJ([DPSOHRID/\VVDYLUXVSRVLWLYH&16VDPSOHGLVSOD\LQJPXOWLSOH VWDLQHGLQFOXVLRQVZLWKLQDQHXURQXVLQJWKH'5,7 a[PDJQLĺFDWLRQ %\FRXUWHV\RI&KDUOHV(5XSSUHFKW/<66$//6&$WODQWD USA Georgia, Direct rapid immunohistochemistry test Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens Discussion 7LVVXHVPD\EHVHOHFWHGIRUWKH'5,7LQWKHVDPHPDQQHUDVWKDWGHVFULEHG in the chapters on brain removal (Chapter 7 DQGWKH')$7 Chapter 11 %HVLGHV removal of the entire CNS or tissue selection via the foramen magnum using the straw technique, the brainstem can be accessed directly by a ventral approach, XVLQJDVFDOSHO )LJ /\VVDYLUXVHVLQWKHEUDLQVRILQIHFWHGDQLPDOVSURGXFH LQWUDF\WRSODVPLF LQFOXVLRQV RI YDULRXV VKDSHV )LJV t 1HJDWLYH VDPSOHV GR QRW KDYH VXFK VSHFLĺF LQFOXVLRQV )LJ $ VLQJOH PLFURVFRSLF ĺHOG may contain numerous round or oval masses and irregular strings of antigen )LJ :KHQ VWDLQHG VSHFLĺFDOO\ ZLWK ELRWLQ\ODWHG DQWLERG\ WKH VXEVWUDWH DPLQR HWK\OFDUED]ROH $(& XSRQR[LGDWLRQIRUPVDURVHUHGHQGSURGXFW DQGPXOWLSOHLQFOXVLRQVPD\EHREVHUYHGZLWKLQLQGLYLGXDOQHXURQV )LJ Haematoxylin counterstain will produce a blue colour to CNS tissue and nuclear EDFNJURXQG )LJ 7KH$(&SURGXFHGSURGXFWLVVXVFHSWLEOHWRGHWHULRUD WLRQLQH[FHVVLYHOLJKWDQGZLOOIDGHLQLQWHQVLW\6WRUDJHRI'5,7VWDLQHGVOLGHVLQ WKHGDUNLVUHFRPPHQGHG 7KH'5,7GHWHFWVWKH1DQWLJHQVRIDOOO\VVDYLUXVHVIRUSULPDU\GLDJQRVLV(LWKHU EURDGO\UHDFWLYHPRQRFORQDORUSRO\FORQDODQWLERGLHVPD\EHFRQMXJDWHGWRELRWLQ IRUXVHLQWKH'5,7$OWHUQDWLYHO\DQLQGLUHFWYHUVLRQRIWKH'5,7PD\EHXWLOL]HG E\XVLQJVXFKSULPDU\XQFRQMXJDWHGDQWLERGLHVIROORZHGE\DQDGGLWLRQDOLQFX EDWLRQRIVHFRQGDU\DQWLERGLHV HJDQWLPRXVHDQWLUDEELWDQWLJRDWHWF,J FRQMXJDWHGWRELRWLQ:KHQXVHGZLWKSDQHOVRIPRQRFORQDODQWLERGLHVFKDUDFWH rization of antigenic variants is also possible (4)&RQMXJDWHVIRUWKH'5,7PD\EH REWDLQHGIURP:+22,(UHIHUHQFHODERUDWRULHVRUWKH\PD\EHVHOISURGXFHGDV described in the chapter on conjugate production )LJ$FFHVVWRWKHEUDLQVWHPRIDGHDG VXVSHFWFDUQLYRUHGLUHFWO\IURPDIXOOWKLFNQHVV ventral approach across the throat latch region XVLQJDVFDOSHORQO\IRUXVHLQWKH'5,7RU ')$7SURWRFROV WKHFDXGDODVSHFWRIWKH carcass is at the top of the image and the ventral cranial aspect of the mandibles is at the ERWWRPSDUWLDOO\FRQFHDOHGE\DJORYHGKDQG Brainstem LD86$ %\FRXUWHV\RI&KDUOHV(5XSSUHFKW/<66$//6&$WODQWD*HRUJ Direct rapid immunohistochemistry test Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens References $QJ0-&/OHQDUHVDV0DQDOR'-DULOOD%7XDVRQ/$YHQLGR('HPHWULD& HWDO(YDOXDWLRQRIGLUHFWUDSLGLPPXQRKLVWRFKHPLFDOWHVW '5,7 RICanis lupus familiaris KLSSRFDPSDOWRXFKLPSUHVVLRQVPHDUVXVLQJDPRQRVSHFLĺFSRO\FORQDO DQWLERG\IRUUDELHVYLUXVGHWHFWLRQ$FWD0HG3KLO t &RHW]HU $ 6DEHWD &70DUNRWWHU : 5XSSUHFKW &( 1HO /+ &RPSDULVRQ RI biotinylated monoclonal and polyclonal antibodies in an evaluation of a direct rapid immunohistochemical test for the routine diagnosis of rabies in southern $IULFD3/R61HJO7URS'LV H GRL MRXUQDOSQWG &RHW]HU$$QDKRU\,'LDV376DEHWD&76FRWW730DUNRWWHU:HWDO(QKDQFHG diagnosis of rabies and molecular evidence for the transboundary spread of the GLVHDVHLQ0R]DPELTXH-6$IU9HW$VVRF HtH GRL MVDYD YL '\HU-/1LH]JRGD02UFLDUL/$ Direct rapid immunohistochemistry test Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens Chapter 13 Immunohistochemistry Introduction Immunohistochemistry utilizes the principle of antigen–antibody reaction to LGHQWLI\ VSHFLĺF DQWLJHQV ZLWKLQ WLVVXH VHFWLRQV 'HWHFWLRQ RI DQWLJHQtDQWLERG\ interactions is achieved by tagging the antibody with a substance that can be YLVXDOL]HG HLWKHU E\ FRQMXJDWLRQ WR D ĻXRUHVFHQW PDUNHU RU HQ]\PH IROORZHG E\FRORXULPHWULFGHWHFWLRQXVLQJĻXRUHVFHLQRUOLJKWPLFURVFRS\7KHWHFKQLTXH RIHPSOR\LQJLPPXQRORJLFDOGHWHFWLRQRIDQWLJHQVGDWHVEDFNWRWKHHDUO\WK FHQWXU\ ZKHQ 0DUUDFN GHPRQVWUDWHG WKDW DQWLW\SKRLG DQG DQWLFKROHUD VHUD ODEHOOHGZLWKGLD]RWL]HGEHQ]LGLQHD]R5VDOWLPSDUWHGDUHGFRORXUWREDFWHULD(1). $OWKRXJKJURXQGEUHDNLQJIRULPPXQRORJLFDOEDVHGGHWHFWLRQRIDQWLJHQV&RRQV determined this labelling method to be relatively insensitive when applied to WLVVXHVDQGVXEVHTXHQWO\GHVFULEHGDVVD\VXWLOL]LQJĻXRUHVFHQWODEHOOHGDQWLER GLHVLQĺ[HGWLVVXHVEXWLQWHUSUHWDWLRQZDVFRQIRXQGHGE\WKHHQKDQFHGHQGRJH QRXVĻXRUHVFHQWDFWLYLW\LQIRUPDOLQĺ[HGWLVVXH(2–6) ,Q 1DNDQHGHVFULEHGDPHWKRGRIDQWLJHQGHWHFWLRQLQWLVVXHXVLQJDQ DQWLERG\FRQMXJDWHGWRDQHQ]\PH KRUVHUDGLVKSHUR[LGDVH DQGXWLOL]HGDFRORX rimetric substrate that could be detected by light microscopy, which is the theo UHWLFDOEDVLVRIPRVWPRGHUQWLVVXHEDVHG,+&DVVD\V(7)7KLVFKDSWHUIRFXVHV RQGHWHFWLRQRIUDELHVYLUXV 5$%9 DQWLJHQVLQIRUPDOLQĺ[HGSDUDIĺQHPEHGGHG ))3( WLVVXHV7KHPDWHULDOVDQGPHWKRGVGHVFULEHDVLQJOHSURWRFRO(8, 9)7KH reader should be aware of, and is encouraged to investigate, the myriad alterna tive nonproprietary and proprietary protocols available (10, 11)7KHGHWHFWLRQRI DQWLJHQVLQ))3(WLVVXHVSUHVHQWVDXQLTXHGLDJQRVWLFFKDOOHQJHIRUWKHYDOLGD tion of the assay and interpretation of the results; the reader unfamiliar with this PHWKRGVKRXOGVHHNLQSXWIURPWHFKQRORJLVWVDQGSDWKRORJLVWVH[SHULHQFHGZLWK DVVD\GHVLJQDQGLQWHUSUHWDWLRQ Materials Reagents s 0DFK$3SRO\PHUV\VWHP %LRFDUH0HGLFDOFDW08/ s 0DFKSUREH s 0DFKSRO\PHU s EDFNJURXQGSXQLVKHU %LRFDUH0HGLFDOFDW%3 0 s 8OWUD&OHDQGLOXHQW 7KHUPR6FLHQWLĺFFDW7$8& Immunohistochemistry Laboratory techniques in rabies Fifth edition Part 3. 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Demonstration of antigens References 0DUUDFN - 1DWXUH RI DQWLERGLHV 1DWXUH t } GRL E &RRQV$+&UHHFK+--RQHV51,PPXQRORJLFDOSURSHUWLHVRIDQDQWLERG\ FRQWDLQLQJ D ĻXRUHVFHQW JURXS 3URF 6RF ([S %LRO 0HG t GRL 3 &RRQV $+ &UHHFK +- -RQHV 51 %HUOLQHU ( 7KH GHPRQVWUDWLRQ RI SQHX PRFRFFDODQWLJHQLQWLVVXHVE\WKHXVHRIĻXRUHVFHQWDQWLERG\-,PPXQRO t &RRQV$+.DSODQ0+/RFDOL]DWLRQRIDQWLJHQLQWLVVXHFHOOV LPSURYHPHQWV LQDPHWKRGIRUWKHGHWHFWLRQRIDQWLJHQE\PHDQVRIĻXRUHVFHQWDQWLERG\-([S 0HG t30,' &RRQV$+6Q\GHU-&&KHHYHU)60XUUD\(6/RFDOL]DWLRQRIDQWLJHQLQWLVVXH FHOOV DQWLJHQVRIULFNHWWVLDHDQGPXPSVYLUXV-([S0HG t .DSODQ0(&RRQV$+'HDQH+:/RFDOL]DWLRQRIDQWLJHQLQWLVVXHFHOOV FHOOXODU GLVWULEXWLRQRISQHXPRFRFFDOSRO\VDFFKDULGHVW\SHV,,DQG,,,LQWKHPRXVH-([S 0HG t 1DNDQH3.3LHUFH*%-U(Q]\PHODEHOHGDQWLERGLHV SUHSDUDWLRQDQGDSSOLFD WLRQIRUWKHORFDOL]DWLRQRIDQWLJHQV-+LVWRFKHP&\WRFKHP t 6ULQLYDVDQ$%XUWRQ(&.XHKQHUW0-5XSSUHFKW&6XWNHU:/.VLD]HN7*HW DO7UDQVPLVVLRQRIUDELHVYLUXVIURPDQRUJDQGRQRUWRIRXUWUDQVSODQWUHFLSLHQWV 1(QJO-0HG tGRL 1(-0RD 0DUWLQHV 5% %KDWQDJDU - GH 2OLYHLUD 5DPRV $0 'DYL +3 ,JOH]LDV 6' .DQDPXUD &7HW DO 3DWKRORJ\ RI FRQJHQLWDO =LND V\QGURPH LQ %UD]LO D FDVH VHULHV/DQFHW t GRL 6 .XPDU */ 5XGEHFN / HGLWRUV ,PPXQRKLVWRFKHPLFDO VWDLQLQJ PHWKRGV WK}HGLWLRQ&DUSLQWHULD 'DNR Immunohistochemistry Laboratory techniques in rabies Fifth edition Part 3. 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Demonstration of antigens s :KHQWKHDQWLJHQLFSURĺOHVZHUHHVWDEOLVKHGGXULQJWKH VWKHOLPLWHG 0$ESDQHOZDVHIIHFWLYHLQWKHLGHQWLĺFDWLRQRIPRUHWKDQ RIWKH FDVHVEHFDXVHGXULQJWKLVSHULRGWKHUHZDVDVLJQLĺFDQWQXPEHURIFDVHV RI FDQLQH UDELHV LQ &HQWUDO DQG 6RXWK $PHULFD 6RPH YLUXVHV LVRODWHG IURP ZLOG DQLPDOV GHVSLWH SUHVHQWLQJ DQ DQWLJHQLF SURĺOH QDPHG E\ PDQ\UHVHDUFKHUVDVqQRQFRPSDWLEOHrUHSHDWHGIUHTXHQWO\ZKHQWKRVH LVRODWHGVDPSOHVZHUHDQDO\VHGLQDUHGXFHG0$ESDQHO)RUH[DPSOH the variant isolated from most of the bats from the species Histiotus velatus SUHVHQWV D SURĺOH ZLWK SRVLWLYH UHDFWLRQ RQO\ IRU 0$E & 7KH 5$%9 isolated from marmosets (Callithrix jacchus) presents a positive reaction SURĺOHRQO\IRU0$EV& DQG& s ,Q%UD]LOWKHZLOGFDQLGYDULDQW Cerdocyon thous SUHVHQWVDSURĺOHVLPLODU WRWKHYDULDQWGHULYLQJIURPGRJV KRZHYHUWKH0$E&GRHVQRWUHDFW WRWKHVHLVRODWHVRUSURGXFHVDZHDNUHDFWLRQ s &OHDUO\WKHUHGXFHGSDQHODOORZVWKHLGHQWLĺFDWLRQRIRWKHU5$%9YDULDQWV EH\RQGWKHLGHQWLĺHGLQLWLDOO\ 7DEOH 7DEOH6WDQGDUGUHDFWLRQSURĺOHVRIGLIIHUHQW5$%9DQWLJHQLFYDULDQWVZLWK0$EV 0RQRFORQDODQWLERGLHV & & & & & & & & $J9 &96(5$6$'3$67 LabL Lab 'RJPRQJRRVH Dog 9DPSLUHEDW Tadarida brasiliensis 9DPSLUHEDW 9HQH]XHOD v v v Lasiurus cinereus v Arizona fox 6NXQNVRXWKFHQWUDO Tadarida brasiliensis 0H[L co) 6NXQN%DMD6& 9DPSLUH RWKHU 7DEOH$QWLJHQLFSURĺOHVHVWDEOLVKHGE\WKHUHGXFHGSDQHORI0$EVIRU5$%9LVRODWHVRIZLOGDQLPDOVLQ%UD]LO 0RQRFORQDODQWLERGLHV & & & & & & & & $J9 &96(5$6$'3$67 Lab Cerdocyon thous " Histiotus velatus " Callithrix jacchus " Antigenic typing by monoclonal antibodies Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens Discussion Antigenic typing methods may be less technically demanding and less costly than molecular techniques and can thus be applied routinely to a large number of cases (9) 7KHVDPSOHVOLGHVFDQEHXVHGDVLPSUHVVLRQVRUSUHSDUHGIURP1$FHOOVRU IURPLQIHFWHGURGHQWEUDLQV8VHVOLGHVSUHSDUHGZLWKQHJDWLYHPRXVHEUDLQDQG EUDLQVRIPLFHLQRFXODWHGZLWKDĺ[HG5$%9DVQHJDWLYHDQGSRVLWLYHSURĺOHV UHVSHFWLYHO\ Slides prepared with original samples of the CNS, stored in good condition, PD\EHXVHGSURYLGHGWKH\VKRZGLVWULEXWLRQRIYLUDODQWLJHQLQtRIWKH ĺHOGVVXUYH\HGDVDVVHVVHGZLWKWKH')$76RPH0$EVPD\SURGXFHYDULDEOH results with original sections prepared from CNS samples, which interfere with the LQWHUSUHWDWLRQDQGGHĺQLWLRQRIWKHDQWLJHQLFSURĺOH 7KHUHFRPPHQGDWLRQLVWKDWERWKVWRFNDQGZRUNLQJ0$EGLOXWLRQVVKRXOGEH prepared using culture medium; however, if this medium is not available, dilu WLRQVFDQEHSUHSDUHGXVLQJEXIIHUHGVDOLQH,IVDOLQHLVXVHGWKHSUHVHUYDWLRQRI WKHVHUHDJHQWVLVDWŗ&IRUWKHVWRFNVROXWLRQDQGEHWZHHQ&DQG&IRU WKHZRUNLQJVROXWLRQ WKHVHWHPSHUDWXUHVPXVWEHULJRURXVO\REVHUYHG5HDJHQWV SUHSDUHGLQEXIIHUHGVDOLQHDUHOHVVVWDEOHDQGPXVWEHSUHSDUHGDWOHDVWPRQWKO\ 6HYHUDO VWXGLHV KDYH EHHQ FRQGXFWHG E\ WKH :+23$+2 &ROODERUDWLQJ &HQWHUVLQDQDWWHPSWWRREWDLQDSDQHORI0$EVZLWKLQFUHDVHGGLVFULPLQDWRU\ capacity (12, 13)&RQVLGHULQJWKHKRVWSRSXODWLRQDQGUHJLRQDOYDULDQWVDSDQHO developed for studies in one area is not necessarily useful for application in other JHRJUDSKLFDODUHDV &DUHPXVWEHWDNHQZKHQSHUIRUPLQJWKHDQWLJHQLFW\SLQJWHVWVZLWK0$EV ,I} WKH UHVXOWV RI LQYROYLQJ D QHZ KRVW VXJJHVW D YDULDQW ZLWK D NQRZQ UHDFWLRQ SURĺOHRUSURYLGHDSURĺOHLQFRPSDWLEOHZLWKWKDWGHĺQHGE\WKHVWDQGDUGSDQHOLW LVUHFRPPHQGHGWKDWWKHUHDFWLRQEHUHSHDWHG Proper preservation by refrigeration or freezing of fresh samples is necessary IRUDQWLJHQLFW\SLQJ $FFXUDWHVSHFLHVLGHQWLĺFDWLRQDQGUHFRUGLQJRIGDWHVDQGORFDWLRQRIFROOHF tion related to each isolate is critical for a meaningful interpretation of resultant GDWD 7KURXJK LWV DSSOLFDWLRQ WR HSLGHPLRORJLFDO UHVHDUFK FODVVLĺFDWLRQ EDVHG RQ 0$EVKDVEURXJKWPDQ\DGYDQFHVWRWKHSUHYHQWLRQDQGFRQWURORIUDELHV$PRQJ WKHPDMRUDGYDQFHVDFKLHYHGE\WKHXVHRI0$EVVHYHUDOGHVHUYHVSHFLDOPHQWLRQ a) The revelation of the diversity of lyssaviruses on the African continent and in Eurasia and their geographical distribution compared to the New World, ZKHUHRQO\5$%9KDVEHHQGRFXPHQWHGZDVEDVHGLQLWLDOO\RQLyssavirus 0$ESDQHOVSURGXFHGE\1RUWK$PHULFDQDQG(XURSHDQUHIHUHQFHODERUD WRULHVZRUNLQJLQFROODERUDWLRQ7KHVHUHVXOWVZHUHFRQĺUPHGPXFKODWHUE\ genetic sequencing (4, 6) Antigenic typing by monoclonal antibodies Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens E 7KHKLJKYDULDELOLW\RI5$%9LQGLIIHUHQWVSHFLHVRIEDWVLQFOXGLQJYDPSLUHV ZDVLGHQWLĺHGLQDODUJHQXPEHURIUHWURVSHFWLYHVXUYH\VFRQGXFWHGLQWKH $PHULFDVZLWKSDQHOVRI0$EV(14–19)7KHVHVWXGLHVHVWDEOLVKHGWKHUHOH YDQFH RI EDWV DV UHVHUYRLUV RI 5$%9 WKURXJKRXW ERWK 1RUWK DQG 6RXWK $PHULFD 6RPH SDQHOV FRQWLQXH WR SUHVHQW OLPLWDWLRQV LQ FKDUDFWHUL]LQJ LVRODWHV RI LQVHFWLYRURXV EDWV ,Q WKHVH FDVHV HLWKHU DGGLWLRQDO 0$EV RU genetic typing should be obtained to identify possible new variants within a species or among species (20) F 'LVWLQFWVSHFLHVRIZLOGFDUQLYRUHVVXFKDVKRDU\IR[HVDQGFUDEHDWLQJ IR[HV ZHUH UHFRJQL]HG DV UHVHUYRLUV RI 5$%9 LQ WKH $PHULFDV (21, 22) These antigenic studies allowed differentiation from variants of canine RULJLQ 2I} IXQGDPHQWDO LPSRUWDQFH DUH WKH PRUSKRPHWULF DQGRU JHQHWLF LGHQWLĺFDWLRQVRIWKHVSHFLHVWRZKLFKDQLVRODWHEHORQJVWRXQGHUVWDQGWKH HSLGHPLRORJLFDOF\FOHVZKLFKHPHUJHDQGSHUSHWXDWH d) The presence of viral spillover from bats to domestic and wild carnivores ZDVUHFRJQL]HGDQGNQRZOHGJHZDVJDLQHGUHJDUGLQJWKHGLVWULEXWLRQRI UDELHV RQ WKH $IULFDQ FRQWLQHQW (XUDVLD DQG WKH $PHULFDV 0RUHRYHU D SRVVLEOH UHVHUYRLU ZDV LGHQWLĺHG LQ D QRQKXPDQ SULPDWH WKH FRPPRQ marmoset (Callithrix jacchus LQ%UD]LO (23):KLOHWKHDQWLJHQLFSURĺOHZDV QRWHVWDEOLVKHGHDVLO\E\DQ\VLQJOH0$ERUSDQHOWKHUHDFWLYLW\SURĺOHZDV PDLQWDLQHGIRUPRUHWKDQ\HDUV(24) e) A Desmodus rotundus YDULDQW $J9 RI YDPSLUH EDWV ZDV LGHQWLĺHG 7KH}UHVHUYRLULVLPSRUWDQWWKURXJKRXW/DWLQ$PHULFDEHFDXVHRIHFRQRPLF ORVVHVLQOLYHVWRFNSURGXFWLRQDQGWKHLULPSDFWRQSXEOLFKHDOWKHVSHFLDOO\ LQWKH$PD]RQ%DVLQDVDWUDQVPLWWHUWRKXPDQV$YDULDQWRIWKHFRPPRQ vampire has also been found in domestic companion animals and wild animals; it is transmitted by Desmodus rotundus or fruit bats of the genus Artibeus, which have shown to host a similar variant (18, 25) I 3DQHOVRI0$EVDQGDQWLJHQLFW\SLQJKDYHEHHQFULWLFDOWRWKHHSLGHPLROR JLFDOLQYHVWLJDWLRQRIDUHDVIUHHRIFDQLQHUDELHVE\YDULDQWVVSHFLĺFWRWKLV species, and also for the use of oral vaccines in the control of wildlife rabies E\XVLQJVXFKUDSLGWHFKQLTXHV J $QWLJHQLFW\SLQJE\0$EVKDVSURYHQXVHIXOWRLGHQWLI\KXPDQUDELHVE\ VHFRQGDU\FURVVVSHFLHVWUDQVPLVVLRQRIEDW!FDW!KXPDQFDVHV(26) ,WKDVEHHQVHHQHVSHFLDOO\LQ/DWLQ$PHULFDQFRXQWULHVVXFKDV%UD]LO(27), Colombia (28) &RVWD 5LFD (29) and Ecuador (30) 7KH ĺUVW FDVHV RI WKLV WUDQVPLVVLRQ F\FOH RFFXUUHG ZLWK LGHQWLĺFDWLRQ RI 9$ $QWLJHQLF 9DULDQW DQGPRUHUHFHQWO\ZLWKDQWLJHQLFYDULDQWVRILQVHFWLYRURXVEDWVLQVRPH FRXQWULHVHVSHFLDOO\LQWKH$QGHDQUHJLRQ h) The number of rabies cases in cats in some regions of the Americas has EHHQJUHDWHUWKDQWKDWLQGRJVDQGLQGLFDWHVWKHULVNUHSUHVHQWHGE\ZLOGOLIH PDNLQJLWPDQGDWRU\WRPRQLWRUWKHVHDQLPDOVDQGSHWVLQUDELHVFRQWUROOHG urban areas, as well as through the practice of rapid diagnostic tests and WKHFRQWLQXLW\RI0$EVXVHGIRUVFUHHQLQJLQFRQWUROSURJUDPPHV(YLGHQWO\ WKHFRXQWULHVRUUHJLRQVWKDWSUHVHQWWKHJUHDWHVWGLIĺFXOWLHVLQODERUDWRU\ diagnosis should have reference laboratories that carry out deeper studies Antigenic typing by monoclonal antibodies Laboratory techniques in rabies Fifth edition Part 3. 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Demonstration of antigens Chapter 15 7SGOHCRCPKFSLKNDKOPSXěGEJNKQTGHOR human rabies antemortem diagnosis Introduction Human rabies may be suspected after the onset of compatible clinical signs DQG FRQĺUPHG E\ GHWHFWLRQ RI DJHQWVSHFLĺF LPPXQRORJLFDO DQG PROHFXODU ELRPDUNHUVXVLQJVHQVLWLYHDQGVSHFLĺFGLDJQRVWLFODERUDWRU\WHVWLQJ$KLVWRU\ of a suspect animal exposure is often a strong indicator of rabies in the differen tial diagnosis of acute progressive encephalitis, but the absence of a bite does not rule out the disease necessarily (1)6SHFLĺFGLDJQRVWLFDOJRULWKPVKDYHEHHQ developed for physicians in selecting priority differentials in encephalitis cases, EXWFRQFRPLWDQWODERUDWRU\GLDJQRVLVUHPDLQVFULWLFDOIRUFRQFOXVLYHLGHQWLĺFDWLRQ of the etiological cause (2) /DERUDWRU\GLDJQRVLVPD\EHFRQGXFWHGDIWHUGHDWKWRFRQĺUPDVXVSHFWFDVH RUZKLOHWKHSHUVRQLVLOO$QWHPRUWHPWHVWLQJPD\DOORZGHĺQLWLYHLGHQWLĺFDWLRQRI SDWKRJHQVLQWKHLOOSDWLHQWZKLOHIDFLOLWDWLQJWKHLGHQWLĺFDWLRQRIRWKHUSHUVRQVZKR PD\KDYHEHHQH[SRVHGWRWKHVDPHVRXUFH$GGLWLRQDOO\HDUO\GLDJQRVLVRIUDELHV is critical for the institution of proper barrier methods to prevent secondary expo sures to excreted virus (in saliva, respiratory secretions and tears for example); IRU} SURSHU ULVN DVVHVVPHQW DQG SURSK\OD[LV RI SDWLHQW FRQWDFWV SV\FKRORJLFDO resolution and closure for family and friends as to etiology, institution and moni toring of experimental therapeutic interventions as appropriate, initiation of grief FRXQVHOOLQJDQGSUHSDUDWLRQVIRUDIDWDODIWHUPDWKRILOOQHVVLIFRQĺUPHG DQGIRU FRQWLQXHGGLDJQRVWLFHYDOXDWLRQLIWKHODERUDWRU\ĺQGLQJVDUHQHJDWLYHDIWHUUREXVW DWWHPSWV %UDLQ WLVVXH LV WKH LGHDO PDWHULDO IRU GLDJQRVLV RI UDELHV LQ WKH ODERUDWRU\ Often, collection of brain biopsy samples for antemortem testing is contraindi FDWHGEHFDXVHRIVHULRXVDQGXQVWDEOHSDWLHQWVWDWXV+RZHYHUVLQFHUDELHVYLUXV 5$%9 DQGRWKHUO\VVDYLUXVHVPRYHFHQWULIXJDOO\DIWHUUHSOLFDWLRQRI&16YLUXV antigens and nucleic acids may be detected in other areas and tissues, such as VDOLYDDQGVNLQ*LYHQWKHXQLTXHSDWKRELRORJ\RIUDELHVDFRPELQDWLRQRIPXOWLSOH WHVWVFRQGXFWHGLQSDUDOOHORQGLIIHUHQWWLVVXHVDPSOHVVXFKDVVHUXP&6)VNLQ biopsy and saliva (and, if safe, to collect also brain biopsy), are necessary to LQFUHDVHVSHFLĺFLW\DQGQHJDWLYHSUHGLFWLYHYDOXHRIUDELHVODERUDWRU\WHVWLQJDQG WRSURYLGHGHĺQLWLYHGLDJQRVLV7KHREMHFWLYHRIWKLVFKDSWHULVWRGHVFULEHDUDSLG VHQVLWLYHDQGVSHFLĺFSURFHGXUHIRUWKHGHWHFWLRQRI5$%9 RURWKHUO\VVDYLUXV DQWLJHQVLQDVNLQELRSV\XVLQJWKHGLUHFWĻXRUHVFHQWDQWLERG\WHVW ')$7 Rapid skin biopsy technique for antemortem diagnosis Laboratory techniques in rabies Fifth edition Part 3. 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Demonstration of antigens Chapter 16 Demonstration of Lyssavirus antigens DXĝOVEXěOMGěRX Introduction 7KH QDWXUH RI LQIHFWLRQ ZLWK UDELHV YLUXV 5$%9 GLFWDWHV D UDSLG VHQVLWLYH VSHFLĺFDQGHIĺFLHQWGLDJQRVLVZKLFKLVRIXWPRVWUHOHYDQFHWRWKHJXLGDQFHRI GHFLVLRQPDNHUVWRDGPLQLVWHU3(3WRWKHSDWLHQWDQGWKHH[HFXWLRQRIHSLGHPLR ORJLFDOPHDVXUHVWRFRQWUROSRWHQWLDOHSLGHPLFV Different techniques are available for diagnosis of rabies in animal and human samples (1, 2)$PRQJWKHWHFKQLTXHVLPPXQRĻXRUHVFHQFHDQGLPPXQRKLVWRFKH PLVWU\ LHWKHLGHQWLĺFDWLRQRIDQWLJHQVDQGLQFOXVLRQVLQWKHVDPSOHV DUHWKHĺUVW choices due to their feasibility and cost, although both methods may present a QXPEHURIIDOVHtQHJDWLYHUHVXOWVWKDWQHFHVVLWDWHWKHXVHRIFRQĺUPDWRU\WHVWV(3) 7KHPRXVHLQRFXODWLRQWHVW 0,7 YLUXVLVRODWLRQLQFHOOFXOWXUHDQGUHYHUVHWUDQV FULSWDVHSRO\PHUDVHFKDLQUHDFWLRQ 573&5 DUHVRPHRIWKHRWKHUWHFKQLTXHV that may be used for this purpose (4, 5) +RZHYHUHWKLFDO LVVXHV DQG WKH WLPH UHTXLUHGWRREWDLQUHVXOWVKDYHJUHDWO\UHGXFHGWKHXVHRI0,72WKHUZLVH573&5 has the advantage of being fast, inexpensive and exhibiting a sensitivity compa UDEOHWRWKDWRIWKH0,7(2–6)0RUHRYHU573&5GRHVQRWDOORZIRUWKHLVRODWLRQRI WKHYLUDOVWUDLQZKLFKFDQEHDFKLHYHGZLWKWHFKQLTXHVWKDWHPSOR\FHOOFXOWXUHV Thus, viral isolation in cell culture has been demonstrated to be an appropriate DOWHUQDWLYHIRUWKHFRQĺUPDWLRQRI5$%9LQIHFWLRQ(2) 6HYHUDOFHOOOLQHVKDYHDOUHDG\EHHQGHVFULEHGDVVXVFHSWLEOHWR5$%9LQIHF WLRQ ZKLFK PDNHV WKHP VXLWDEOH IRU XVH LQ GLDJQRVLV (7–10) +RZHYHULVRODWLRQ RI 5$%9 LQ FHOO FXOWXUH XVXDOO\ UHTXLUHV D IHZ GD\V t GD\V WR DOORZ IRU WKH GHWHFWLRQRIYLUDODQWLJHQVE\LPPXQRĻXRUHVFHQFHRUDQ\RWKHUWHFKQLTXH(2, 4) )OXRUHVFHQFHDFWLYDWHGFHOOVRUWLQJ )$&6 LVDQDXWRPDWHGWHFKQLTXHWKDWLVXVHG for the analyses of cells in suspension (11)7KLVPHWKRGFDQVXEVWDQWLDOO\UHGXFH the time required for diagnosis (12). Through a system using laser beams, optical PHDQVVRIWZDUHDQGĻXRUHVFHQWG\HVWKLVWHFKQLTXHDOORZVIRUWKHGHWHFWLRQDQG TXDQWLĺFDWLRQRIPXOWLSOHDQWLJHQVLQFHOOV7KLVWHFKQLTXHLVIDVWHDV\WRSHUIRUP and reproducible, and it allows for the simultaneous analyses of many samples, ZKLFK UHSUHVHQWV DQ LPSRUWDQW DGYDQWDJH IRU URXWLQH ODERUDWRULHV $GGLWLRQDOO\ EHFDXVHWKHDQDO\VLVRIWKHĻXRUHVFHQFHLVDXWRPDWHGWKHLQWHUKXPDQYDULDWLRQ LQWKHUHVXOWVLVGUDVWLFDOO\UHGXFHG 7KH XVH RI )$&6 LQ YLURORJ\ LV ZHOO HVWDEOLVKHG DQG WKH TXDQWLĺFDWLRQV RI infected cells or viral antigens in infected cells have already been shown for several YLUXVHVLQFOXGLQJGHQJXHURWDYLUXVDQG+,9(13–18))$&6DOORZVWKHGHWHFWLRQDQG TXDQWLĺFDWLRQ RI 5$%9 DQWLJHQV LQ LQIHFWHG FHOOV XVLQJ D ODVHU WKDW H[FLWHV WKH ĻXRURSKRUHFRQMXJDWHGWRWKH5$%9DQWLERG\DQGWKXVHQDEOHVWKHGHWHFWLRQRI GLIIHUHQW5$%9DQWLJHQVVXFKDVULERQXFOHRSURWHLQVRUJO\FRSURWHLQVGHSHQGLQJ #DLNMRSQ@SHNMNE+XRR@UHQTR@MSHFDMRAXkNVBXSNLDSQX Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens RQWKHVSHFLĺFLW\RIWKHFRQMXJDWHXVHG(12, 19))$&6KDVDGGLWLRQDOO\EHHQXVHG IRUWKHGHWHFWLRQDQGTXDQWLĺFDWLRQRI5$%9QHXWUDOL]LQJDQWLERGLHV(20, 21) A protocol similar to that described below can be easily adapted to perform WKHTXDQWLĺFDWLRQRIDQWLERGLHVE\VLPSO\DGGLQJVRPHVWHSVDQGXVLQJDFRQWURO VHUXP ZLWK D SUHGHĺQHG QHXWUDOL]LQJ DQWLERG\ WLWUH 5HFHQWO\ D 9HUR FHOO OLQH H[SUHVVLQJWKH5$%9JO\FRSURWHLQ * RQWKHFHOOPHPEUDQHZDVXVHGDVDQDOWHU QDWLYHPHWKRGIRUWKHGHWHFWLRQDQGTXDQWLĺFDWLRQRIDQWLERGLHVWDUJHWLQJWKH* SURWHLQZLWKWKHDGYDQWDJHRIQRWUHTXLULQJWKHKDQGOLQJRIWKHLQIHFWLRXV5$%9(19) %HFDXVHWKH)$&6UHGXFHVWKHWLPHUHTXLUHGIRUDFRQĺUPDWRU\UDELHVGLDJQRVLV the routine use of this technique could help to guide both medical and epidemio logical measurements, prevent the use of unnecessary human vaccinations and KHOSSXEOLFKHDOWKDXWKRULWLHVLQWKHHVWDEOLVKPHQWRIDFWLRQVWRDYRLGHSLGHPLFV Protocol 7KH GHWHFWLRQ RI O\VVDYLUXV DQWLJHQV LQ FHOO FXOWXUHV E\ )$&6 LV VLPLODU WR D WUDGLWLRQDOLPPXQRĻXRUHVFHQFHDVVD\XVLQJĻXRUHVFHQFHPLFURVFRS\ %ULHĻ\ 7KHFHOOVDUHLQIHFWHGZLWKDVDPSOHRIDYLUXVVWRFNRUDVXVSHQVLRQRI tissue samples suspected of infection and, after a period of incubation, LQWUDFHOOXODUVWDLQLQJIRUWKHYLUXVDQWLJHQVLVSHUIRUPHG 'LIIHUHQW FHOOV OLQHV VXFK DV %+. & $7&& &&/ & $7&& &&/ DQGPXULQHQHXUREODVWRPDFHOOV 01$ KDYHDOUHDG\EHHQXVHG IRUUDELHVYLUXVGHWHFWLRQE\)$&6(12, 19) %+. DQG & FHOO OLQHV ZHUH FXOWXUHG LQ 'XOEHFFRpV PRGLĺHG (DJOHpV PHGLXP ) 6LJPD$OGULFK VXSSOHPHQWHG ZLWK JOXWDPLQH PPRO 7KHUPR )LVKHU IHWDO FDOI VHUXP 6LJPD$OGULFK SHQLFLOOLQVWUHS WRP\FLQ ,8ŻJP/ 7KHUPR )LVKHU DQG DPSKRWHULFLQ % ŻJ SHU}P/ 7RGHWHUPLQHWKHVHQVLWLYLW\RIWKHWHFKQLTXHLWLVLPSHUDWLYHWKDWWKHNLQH tics of infection be examined for the selected cell line, preferentially using DODERUDWRU\VWUDLQ LQRXUFDVH3DVWHXUYLUXV39 DQGDVWUHHWUDELHVYLUXV :56 LQRXUFDVHRQHLVRODWHGIURPDQDWXUDOO\LQIHFWHGFRZ 7KHFHOOVZHUHSODWHGLQZHOOGLVKHVRYHUQLJKW [FHOOVZHOO DQG LQIHFWHGZLWK&&,'RI39RU:56IRUKDW&LQD&2 atmos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kNVBXSNLDSQX Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens SURWHLQSRO\FORQDO),7&FRQMXJDWHGDQWLERGLHVLQ3%6IRUPLQDW&LQ WKH}GDUN7KHFHOOVZHUHWKHQZDVKHGDJDLQUHFRYHUHGLQ&RXOWHU,VRWRQ,, DQGVWRUHGDW&LQWKHGDUNXQWLOĻRZF\WRPHWULFDQDO\VLV(12) )LJ+LVWRJUDPVVKRZLQJWKHNLQHWLFVRIWKHLQIHFWLRQRIUDWJOLRPDFHOOV & E\DUDELHVYLUXVLVRODWHGIURPDQDWXUDOO\LQIHFWHG FRZ7KHFHOOVZHUHLQIHFWHGZLWK&&,'RIWKHVDPSOHDQGWKHTXDQWLĺFDWLRQRIWKH&LQIHFWHGFHOOVZDVSHUIRUPHGZLWKWKHĻRZ F\WRPHWU\GHVFULEHGLQWKHSURWRFRO$),7&FRQMXJDWHGDQWLUDELHVYLUXVQXFOHRSURWHLQ SRO\FORQDODQWLERG\ ZDVXVHGIRUDQWLJHQ GHWHFWLRQLQD)$&6&DOLEXU(12) The protocol described herein has proven effective for detection of intracellular 5$%9DQWLJHQLQWKH%+.DQG&FHOOOLQHV+RZHYHUVRPHFKDQJHVDQGQHZ commercial reagents that have lately become available could improve the tech QLTXHDQGUHVXOWV)RUH[DPSOHLQWKHSURWRFROGHVFULEHGIRUWKHGHWHFWLRQRI5$%9 DQWLJHQVLQ01$FHOOVWKHDXWKRUVRIWKHVWXG\DGRSWHGVRPHFKDQJHVWKDWFRXOGEH DGYDQWDJHRXV 7KHQXPEHURIFHOOVZDVDGMXVWHGWR[FHOOVP/EHIRUH VWDLQLQJDQGWKHFHOOVZHUHZDVKHGWZLFHZLWKFROG3%6 }UPLQIRUPLQDW }& 7KHFHOOVZHUHWKHQLQFXEDWHGIRUPLQDW&ZLWKŻ/RI&\WRĺ[ &\WRSHUPVROXWLRQ %'%LRVFLHQFHV86$ $IWHUQHZZDVKLQJV WZLFH LQWKHVWDL QLQJVROXWLRQ ERYLQHVHUXPDOEXPLQVRGLXPD]LGHDQGVDSRQLQ LQ3%6 WKHFHOOVZHUHLQFXEDWHGZLWK5$%9DQWLQXFOHRFDSVLGDQWLERG\WKDWZDV ),7&FRQMXJDWHG LQWKHVWDLQLQJVROXWLRQ IRUPLQLQWKHGDUN)LQDOO\WKHFHOOV ZHUHZDVKHGDJDLQDQGWKHĺQDOSHOOHWZDVVXVSHQGHGLQ}Ż/RI)$&6ĻRZ EXIIHU %'%LRVFLHQFHV DQGVWRUHGDW&XQWLOĻRZF\WRPHWU\ZDVSHUIRUPHG :KHQ WKH VDPSOHV ZHUH WR EH DQDO\VHG LPPHGLDWHO\ 3%6 FRXOG EH XVHG WR UHFRYHUWKHFHOOSHOOHWV2WKHUZLVHZKHQWKH)$&6DQDO\VHVZHUHSHUIRUPHGODWHU tGD\V DEXIIHUFRQWDLQLQJSIRUPDOGHK\GHZDVXVHGDQGWKHVDPSOHV QHHGHGWREHSURWHFWHGIURPOLJKWDQGPDLQWDLQHGDW& 5HVHDUFKHUVLQWHUHVWHGLQWKHXVHRI)$&6WRGHWHFWDQGTXDQWLI\LQWUDFHOOXODU lyssavirus antigens should always be aware that the sensitivity to the virus differs not only between different cell lineages but also with the number of passages of each lineage (7, 8, 10, 22) 6WDQGDUGL]DWLRQ LQ ORFDO FRQGLWLRQV LV DGYLVDEOH #DLNMRSQ@SHNMNE+XRR@UHQTR@MSHFDMRAXkNVBXSNLDSQX Laboratory techniques in rabies Fifth edition Part 3. 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Equipment, reagents and supplies • Equipment. 6HYHUDOFRPSDQLHVRIIHUĻRZF\WRPHWHUVLQFOXGLQJ%' ZZZ EGFRP %LR5DG ZZZELRUDGFRP 7KHUPR)LVKHU6FLHQWLĺF ZZZWKHU PRĺVKHUFRP DQG%HFNPDQ&RXOWHU ZZZEHFNPDQFRP • Software. )RUWKHDQDO\VLVRIWKH)$&6GDWDWKHHTXLSPHQWKDGDYDLODEOH VRIWZDUH)RUH[DPSOHWKH%'%LRVFLHQFHVĻRZF\WRPHWHUVKDYHVRIWZDUH IRUDQDO\VHVVXFKDVWKH&HOO4XHVWRQWKH)$&6&DOLEXUDQGPRVWUHFHQWO\ WKH%')$&6'LYD70$GGLWLRQDOO\VRPHVRIWZDUHLVDYDLODEOHIUHHRIFKDUJH on the internet; see the Purdue University Cytometry Laboratories catalog for examples (KWWS ZZZF\WRSXUGXHHGXĻRZF\WVRIWZDUH&DWDORJKWP • Reagents. )RUFHOOĺ[DWLRQDQGSHUPHDELOL]DWLRQWKHXVHRI&\WRĺ[&\WRSHUP VROXWLRQ %'%LRVFLHQFHV86$ FDWDORJQXPEHU LVUHFRPPHQGHG The intracellular staining of the cells with antibodies can be performed with WKHSHUPDQGZDVKVROXWLRQSURYLGHGLQWKH&\WRĺ[&\WRSHUPNLW5HDJHQWV DQWLERG\WLWUDWLRQUHVHDUFKFDQXVHWKH%'70&RPS%HDGV %'%LRVFLHQFHV 86$ FDWDORJQXPEHU #DLNMRSQ@SHNMNE+XRR@UHQTR@MSHFDMRAXkNVBXSNLDSQX Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens Chapter 17 Rapid immunochromatographic tests for the detection of rabies virus antigens in brain material Introduction 'HFHQWUDOL]HGRUSRLQWRIFDUHWHVWLQJKDVWKHSRWHQWLDOWRLPSURYHWKHVXUYHLO ODQFHRIPDQ\KXPDQDQGDQLPDOGLVHDVHVLQORZUHVRXUFHVHWWLQJV(1)$PRQJ WKRVHWHVWVWKDWFDQEHXVHGXQGHUĺHOGFRQGLWLRQVODWHUDOĻRZGHYLFHV /)'V t DOVRUHIHUUHGWRDVUDSLGLPPXQRFKURPDWRJUDSKLFGLDJQRVWLFWHVWV 5,'7V ODWHUDO ĻRZ LPPXQRDVVD\V /)$V RU LPPXQRFKURPDWRJUDSKLF VWULS WHVWV t SUHVHQW LQWHUHVWLQJFKDUDFWHULVWLFV7KH\DUHUDSLGDQGHDV\WRXVHZLWKRXWVSHFLDOWUDL QLQJ QHHGHG IRU LPSOHPHQWDWLRQ DQG HYDOXDWLRQ $QRWKHU DGYDQWDJH LV WKDW WKH tests have no special storage requirements in terms of temperature; they can EHVKLSSHGDQGVWRUHGDWURRPWHPSHUDWXUH/)'VZHUHLQLWLDOO\GHVFULEHGLQWKH VDQGWKHĺUVWFRPPHUFLDOO\DYDLODEOH/)'LQ ZDVDKRPHSUHJQDQF\ test (2)7KHUHDIWHUWKLVWHVWPHWKRGZDVLQWURGXFHGLQWRPDQ\GLIIHUHQWDUHDV(3). 7KLVH[SDQVLRQLQFOXGHGWKHGLDJQRVLVRIYLUDOGLVHDVHVVXFKDVIRRWDQGPRXWK disease (4)DYLDQLQĻXHQ]D(5) and Ebola virus disease (6) 7KH/)'VIRUGHWHFWLRQRI5$%9ZHUHGHYHORSHGDQGSURRIRISULQFLSOHVWXGLHV \LHOGHG HQFRXUDJLQJ UHVXOWV UHJDUGLQJ VHQVLWLYLW\ DQG VSHFLĺFLW\ (7, 8) )XUWKHU VWXGLHVFRQFHQWUDWHGRQWKH$QLJHQ%LR1RWH/)'VKRZLQJLWVSRWHQWLDOWRGHWHFW O\VVDYLUXVHVHVVHQWLDOO\IURP$IULFDWKH0LGGOH(DVW$VLDDQG(XURSH5HSRUWHG VHQVLWLYLWLHV FRPSDUHG ZLWK WKH ')$7 UDQJHG EHWZHHQ DQG LQ ĺHOG samples (9–13)+RZHYHULQDFRPSDUDWLYHVWXG\DVVHVVLQJWKHGLDJQRVWLFVHQVLWL YLW\RIVL[GLIIHUHQW/)'VVWULFWO\IROORZLQJPDQXIDFWXUHUVpLQVWUXFWLRQVLQVXIĺFLHQW test characteristics prompted a careful consideration on the selection and use of /)'VIRUGHWHFWLRQRI5$%9(14)7KLVFKDSWHUVXPPDUL]HVWKHJHQHUDOSULQFLSOH alongside the various components and discusses the use, limitations and further QHHGVIRULPSURYHPHQW Principle 2QFHDOLTXLGVDPSOHLVDSSOLHGWRWKHVDPSOHSDGJROGFRQMXJDWHGDQWLERGLHV ELQGVSHFLĺFDOO\WRWKHDQWLJHQVLQWKHVDPSOH7KLVDQWLJHQtDQWLERG\FRPSOH[ then travels along a nitrocellulose membrane by liquid migration and is eventually LPPRELOL]HGE\DVHFRQGDQWLERG\ZKLFKLVĺ[HGRQWKHWHVWVWULS(15)7KLVDJJUH JDWLRQRIWKHFRQMXJDWHIRUPVDYLVLEOHOLQHLQWKHWHVWDUHD8QERXQGFRQMXJDWHG antibodies are later captured downstream at a second line representing the control OLQH )LJ ([FHVVUHDJHQWVPRYHSDVWWKHFDSWXUHOLQHVDQGDUHHQWUDSSHGLQ WKHZLFNRUDEVRUEHQWSDG8VXDOO\WKHWHVWLVUHDGYLVXDOO\DIWHUt}PLQDQGLV YDOLGLIWKHFRQWUROOLQHDSSHDUV Rapid immunochromatographic tests Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens ) ) %! ) ) ") ) # ) ) ) ' ) ! %) ! " )( ) ) )$ # ) ! ) ! 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Demonstration of antigens References $OWHODDU$)0XQR]-+HFN$-1H[WJHQHUDWLRQSURWHRPLFV WRZDUGVDQLQWH JUDWLYHYLHZRISURWHRPHG\QDPLFV1DW5HY*HQHW tGRL QUJ 0DQQ 0 .XODN 1$ 1DJDUDM 1 &R[ - 7KH FRPLQJ DJH RI FRPSOHWH DFFX UDWH DQG XELTXLWRXV SURWHRPHV 0RO &HOO t GRL M PROFHO +DQ ; $VODQLDQ $ Mass spectrometry-based proteomic approaches Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens .DW],6'LDV0+/LPD,)&KDYHV/%5LEHLUR2*6FKHIIHU.&HWDO/DUJH SURWHLQDVDSRWHQWLDOWDUJHWIRUXVHLQUDELHVGLDJQRVWLF$FWD9LURO GRL DYBB 5DP\D50RKDQD6XEUDPDQLDQ%6LYDNXPDU96HQWKLONXPDU5/6DPEDVLYD 5DR.56ULQLYDVDQ9$([SUHVVLRQDQGVROXELOL]DWLRQRILQVHFWFHOOEDVHGUDELHV YLUXVJO\FRSURWHLQDQGDVVHVVPHQWRILWVLPPXQRJHQLFLW\DQGSURWHFWLYHHIĺFDF\ LQPLFH&OLQ9DFFLQH,PPXQRO t GRL &9, /DHPPOL8.&OHDYDJHRIVWUXFWXUDOSURWHLQVGXULQJWKHDVVHPEO\RIWKHKHDG RIEDFWHULRSKDJH71DWXUH t Mass spectrometry-based proteomic approaches Laboratory techniques in rabies Fifth edition Part 3. Demonstration of antigens #NNGW2RGPCRCěKONOHDTėGRSCNFRGCIGNěS [/DHPPOLVDPSOHEXIIHUS+ 6'6 JO\FHURO PHUFDSWRHWKDQRO EURPRSKHQROEOXH PRO7ULV+&O Dissolve in ddHO Polyacrylamide solution 6ROXWLRQ$ DFU\ODPLGHDQGELVDFU\ODPLGHGLVVROYHGLQZDWHU 3RO\DFU\ODPLGHVROXWLRQ P/ Solution A (mL) 6ROXWLRQ% P/ ddHO DPPRQLXPSHUVXOSKDWH / 7(0(' / 6ROXWLRQ% 7ULV%DVHPRO S+ DQG6'6GLVVROYHGLQZDWHU $GGHGMXVWEHIRUHSRXULQJJHO Running buffer J7ULVEDVH Q0 JJO\FLQH PPRO J6'6 %ULQJWKHWRWDOYROXPHXSWR/ZLWKGG+O )L[LQJVROXWLRQ PHWKDQRO JODFLDODFHWLFDFLG Staining solution &RRPDVVLHEULOOLDQWEOXH PHWKDQRO JODFLDODFHWLFDFLG Destaining solution PHWKDQRO JODFLDODFHWLFDFLG Mass spectrometry-based proteomic approaches Laboratory techniques in rabies Fifth edition Part 3. 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Demonstration of viral antibodies Part 4. Demonstration of viral antibodies Laboratory techniques in rabies Fifth edition Part 4. Demonstration of viral antibodies Chapter 19 6JGRCPKFĝTORGSEGNěHOETSKNJKDKěKONěGSě Introduction 7KHFODVVLFDOUDSLGĻXRUHVFHQWIRFXVLQKLELWLRQWHVW 5)),7 ZDVGHYHORSHGDW the Communicable Disease Center (now the United States Centers for Disease &RQWURODQG3UHYHQWLRQ>&'&@ LQ E\-6PLWK3 1@OHCkTNQDRBDMSENBTRHMGHAHSHNMSDRS Laboratory techniques in rabies Fifth edition Part 4. 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Demonstration of viral antibodies )RUWKHYLUXVEDFNWLWUDWLRQ/LVDGGHGLQWKHĺUVWIRXUZHOOV +t+ DQGD VHULDOGLOXWLRQLVSHUIRUPHGZLWKDGLOXWLRQVWHSRIIRXU WUDQVIHURI/IURPURZ WRURZ )RUWKHODVWOLQHRIZHOOVWKH/DUHGLVFDUGHGDQGWKHĺQDOYROXPHLQ HDFKZHOOLVHTXDOWR/ Incubation of the plates 7KHPLFURSODWHVDUHLQFXEDWHGIRUKDW&&&2 Distribution of the cell suspension 7KHFHOOVDUHPDLQWDLQHGLQ*0(0VXSSOHPHQWHGZLWK)&6DQGDQWLELR WLFV)RUWKH)$91WHVWDVXEFRQĻXHQWFXOWXUHRI%+.FHOOVDJHGGD\VVKRXOG EHWU\SVLQL]HG7KHFRQFHQWUDWLRQRIFHOOVVKRXOGEHFRXQWHGE\XVLQJDFHOOFRXQ WLQJ FKDPEHU IRU H[DPSOH D KDHPRF\WRPHWHU >1HXEDXHU FKDPEHU@ 7KH ĺQDO FHOOFRQFHQWUDWLRQPXVWEHFDOFXODWHGIROORZLQJWKHPDQXIDFWXUHUpVLQVWUXFWLRQV GHSHQGLQJRQWKHW\SHRIFRXQWLQJFKDPEHUXVHG7KHFRQFHQWUDWLRQRIFHOOVLV H[SUHVVHGLQFHOOVP/ $FFRUGLQJWRWKHQXPEHURIFHOOVFRXQWHGWKHFHOOVDUHUHVXVSHQGHGWRREWDLQ DĺQDOFRQFHQWUDWLRQRI[FHOOVP/XVLQJ'0(0VXSSOHPHQWHGZLWK})&6 DQGDQWLELRWLFVDVGLOXHQWDQGWKHQ/RIWKHFHOOVXVSHQVLRQLVGLVWULEXWHGWR HDFKZHOO Incubation of the plates 7KHPLFURSODWHVDUHLQFXEDWHGIRUDSSUR[LPDWHO\KDW&&&2 )L[DWLRQ $IWHUKRILQFXEDWLRQWKHPHGLXPLVGLVFDUGHG7KHPLFURSODWHVDUHULQVHG ĺUVW LQ 3%6 EXIIHU WKHQ LQ DFHWRQH 7KH PLFURSODWHV DUH ĺ[HG ZLWK DFHWRQHDWURRPWHPSHUDWXUHIRUPLQLQDIXPHKRRG7KHDFHWRQHLVGLVFDUGHG DQGWKHPLFURSODWHVDUHDLUGULHGDWURRPWHPSHUDWXUH $OO WKH SODWHV DUH FKHFNHG IRU SRVVLEOH F\WRWR[LFLW\ ODFN RI PRQROD\HU FHOO IUHHSDWFKHVLQWKHPRQROD\HUGHWDFKPHQWRIPRQROD\HUV 7KHREVHUYDWLRQVDUH ZULWWHQRQWKHUDZUHDGLQJVKHHW Staining 6RPH/RIWKH),7&FRQMXJDWH GLOXWHGDSSURSULDWHO\LQ3%6 DUHGLVWULEXWHG LQHDFKZHOORIWKHSODWHV7KHPLFURSODWHVDUHJHQWO\URFNHGWRHQVXUHJRRGGLVWUL EXWLRQLQWKHHQWLUHVXUIDFHRIWKHZHOO7KHPLFURSODWHVDUHLQFXEDWHGIRUPLQ DW&WKHQWKHĻXRUHVFHQWFRQMXJDWHLVGLVFDUGHGDQGWKHPLFURSODWHVDUH ULQVHGWZLFHZLWK3%6EXIIHU7KHPLFURSODWHVDUHLQYHUWHGEULHĻ\RQDEVRUEHQW SDSHUWRUHPRYHWKHUHVLGXDO3%6 Fluorescent antibody virus neutralization test Laboratory techniques in rabies Fifth edition Part 4. Demonstration of viral antibodies Reading 7KHZHOOVDUHREVHUYHGWKRURXJKO\7KHUHDGLQJLVTXDOLWDWLYHDQGWKLVLVDQ qDOO}RUQRWKLQJrUHDGLQJ ,IWKHUHLVRQHRUVHYHUDOĻXRUHVFHQWFHOOVWKHZHOOLVFRQVLGHUHGDVSRVLWLYH LI}WKHUHLVQRĻXRUHVFHQWFHOOWKHZHOOLVFRQVLGHUHGDVQHJDWLYH7KHFRQWUROSODWH VKRXOGEHUHDGĺUVW Calculation of the titres The logDWLWUHVRIWKH&96WLWUDWLRQWKHQDLYHUHIHUHQFHVHUXPWKHSRVLWLYH reference serum and the test sera are calculated according to the Spearman– .ÁUEHUPHWKRG Where: s ORJ GLOXWLRQ ORJ' ORJDULWKPRIWKHGLOXWLRQZKHUHRIWKH SRVLWLYHZHOOVLVIRXQG WKDWLVRIWKHĻXRUHVFHQFH s [ t ORJRIWKHVPDOOHVWGLOXWLRQZLWKDOOQHJDWLYHZHOOV s G ORJRIWKHGLOXWLRQVWHS s UL QXPEHURIQHJDWLYHZHOOV DQG s QL QXPEHURIUHSOLFDWHV For the sera: logDRIWKHVHUXP > QXPEHURIQHJDWLYHZHOOV [ORJRIGLOXWLRQVWHSt ORJ RIGLOXWLRQVWHS ORJRIWKHORZHVWGLOXWLRQZKHUHWKHUHDUHIRXUQHJDWLYHZHOOV@ And for the CVS virus: logDRIWKHYLUXV > QXPEHURISRVLWLYHZHOOV [ORJRIGLOXWLRQVWHSt ORJ RIGLOXWLRQVWHS ORJRIWKHORZHVWGLOXWLRQZKHUHWKHUHDUHIRXUSRVLWLYHZHOOV@ Conversion of the titres The conversion of the logDWLWUHRIWKHVHUXPLQWKH,8P/WLWUHLVSHUIRUPHG by using the following formula: )RUWKHWKHRUHWLFDOORJ'RISRVLWLYHUHIHUHQFHVHUXPWZRNLQGVRIYDOXHVFDQ be used to convert the logDWLWUHLQ,8P/ HLWKHUWKHqYDOXHRIWKHGD\rRUWKH mean logDYDOXHVRISRVLWLYHUHIHUHQFHVHUXP Fluorescent antibody virus neutralization test Laboratory techniques in rabies Fifth edition Part 4. Demonstration of viral antibodies Validation of the test 7KHUHVXOWVRIWLWUDWLRQRI&96QDLYHVHUXPDQGSRVLWLYHVHUXPDUHUHSRUWHGRQ DFRQWUROFDUGIRUHDFKRIWKHVHWKUHHFRQWUROV (YHU\SHUVRQZKRSHUIRUPVWKH)$91WHVWLQWKHODERUDWRU\VKRXOGKDYHKLVRU KHURZQFRQWUROFDUGIRU&96YLUXVDQGQDLYHDQGSRVLWLYHUHIHUHQFHVHUD7KH FRQWUROWHVWUHVXOWVRIWKH)$91WHVWDUHFRPSDUHGZLWKWKHDFFXPXODWHGFRQWURO WHVWUHVXOWVIURPSUHYLRXV)$91WHVWVRIWKHWHFKQLFLDQ V The test is validated only if the current values of the three controls are not statis WLFDOO\GLIIHUHQWIURPWKHPHDQVWDQGDUGGHYLDWLRQ 6' RIDOOYDOXHVREWDLQHG LQWKHSUHYLRXVWHVWV,IRQHRIWKHFRQWUROVLVVWDWLVWLFDOO\GLIIHUHQWIURPWKHPHDQ }}6'WKHWHVWLVQRWYDOLGDWHGDQGWKHVHUDPXVWEHWLWUDWHGRQFHDJDLQ Discussion 7KH)$91WHVWKDVEHHQUHFRPPHQGHGE\WKH2,(VLQFH (13) and by WHO VLQFH (10) (XURSHDQ &RPPLVVLRQ 5HJXODWLRQ (& 1R (14) also UHTXLUHVPHDVXULQJ91$DVDSURRIRIYDFFLQDWLRQDJDLQVWUDELHVWRDOORZWKHIUHH movement of pets within the European Union and between certain developing DQG(XURSHDQFRXQWULHV$VVXFKWKH)$91WHVWLVWKHPRVWZLGHO\XVHGE\WKH approved rabies serology laboratories (KWWS HFHXURSDHXIRRGDQLPDOOLYHDQL PDOVSHWVDSSURYDOBHQKWP LQWKHFRQWH[WRILQWHUQDWLRQDOWUDGH,QGHHGRI these laboratories used this serum neutralization test to perform rabies serological FRQWUROVRQGRPHVWLFFDUQLYRUHV 7KH)$91WHVWLVDOVRXVHGRQZLOGOLIHVDPSOHVWRHYDOXDWHWKHLPSDFWRIRUDO vaccination campaigns (15) +RZHYHU ĺHOG VDPSOHV DUH RIWHQ RI SRRU TXDOLW\ (haemolysis, bacterial contamination) (16) DQG FHOOV XVHG LQ WKH )$91 WHVW DUH sensitive to any cytotoxic products and contaminating agents present in samples, rendering it impossible to obtain reliable results (17, 18) and leading to inadequate PRQLWRULQJRIWKHHIIHFWLYHQHVVRIRUDOYDFFLQDWLRQFDPSDLJQV7RRYHUFRPHWKH F\WRWR[LFLW\SUREOHPRIĺHOGVDPSOHVVRPHPRGLĺHG)$91WHVWVZHUHGHYHORSHG (19, 20)7KH)$91WHVWLVDOVRZLGHO\XVHGLQVHURSUHYDOHQFHVWXGLHVIRUYDFFLQDWHG dog and cat populations (21) in serological surveys in wild and domestic animals as well as in immunogenicity studies of animal or human vaccines (more than }FLWDWLRQV Critical parameters As described in the OIE manual (11), some steps of this procedure may be PRGLĺHGWRFRPSO\ZLWKWKHKHDOWKDQGVDIHW\UHTXLUHPHQWVDQGLQGLYLGXDOZRUNLQJ practices of the laboratory, but others must not be changed as they are standard RSHUDWLQJSURFHGXUHV The following steps must not be changed for the virus titration: s ,QRFXODWLRQRIDKPRQROD\HU s IROGGLOXWLRQVSUHSDUHGXVLQJ P/RIGLOXHQWDQGP/RIYLUXVVXVSHQVLRQ Fluorescent antibody virus neutralization test Laboratory techniques in rabies Fifth edition Part 4. 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Demonstration of viral antibodies Chapter 23 Demonstration of lyssavirus antibodies by pseudotype virus micro-neutralization assays Introduction 3VHXGRW\SHGYLUXVHV 379 DUHGHĺQHGDVYLUDOSDUWLFOHVZKLFKFRQVLVWRIDFRUH (matrix, capsid, nucleocapsid) originating from one virus, which is surrounded by D OLSLG HQYHORSH FRPSULVLQJ D YLUDO HQYHORSH SURWHLQ 9(3 SURWUXGLQJ IURP WKH RXWHU VXUIDFH ,Q PRVW FDVHV WKH JHQRPH KDV EHHQ HQJLQHHUHG WR LQFRUSRUDWH a reporter gene while the genetic elements required for replication have been UHPRYHG7KLV}RIIHUVDVDIHV\VWHPIURPZKLFKWRVWXG\SURSHUWLHVUHODWHGWR9(3 ZLWK379DEOHWRDFWDVVXUURJDWHVWRKLJKO\SDWKRJHQLFYLUXVHVVXFKDVUDELHV YLUXV 5$%9 IRUVHURORJLFDODQGYLUXVHQWU\VWXGLHV8SRQ379WUDQVGXFWLRQRI VXVFHSWLEOHFHOOVGHWHFWLRQRIUHSRUWHUJHQHH[SUHVVLRQFDQEHXVHGWRLQIHU9(3 LQWHUDFWLRQZLWKDQWLERGLHVDQGFHOOUHFHSWRUVZKLFKFDQEHHDVLO\DQGTXLFNO\ TXDQWLĺHGWRGHWHUPLQHWKHQHXWUDOL]LQJSRWHQF\RIVHURORJLFDOVDPSOHV 5HWURYLUXVHV KDYH ZLGHO\ EHHQ XVHG DV FRUHV IRU 379DQG FXUUHQW SURGXF WLRQSURWRFROVDUHEDVHGRQVHYHUDOGHFDGHVRIUHWURYLUDOYHFWRUGHYHORSPHQW}(1) 7KLV} GHYHORSPHQW LV FORVHO\ DOOLHG WR WKH JHQH WKHUDS\ ĺHOG ZKHUH UHWURYLUDO vectors are favoured due to the ability to stably integrate transfer genes into the cell genome without transferring viral genes (2, 3) (DUO\ UHWURYLUDO YHFWRUV ZHUH EDVHGRQJDPPDUHWURYLUXVHVRIWHQPXULQHOHXNDHPLDYLUXV 0/9 +RZHYHUWKHLU XWLOLW\ZDVUHVWULFWHGE\RQO\EHLQJDEOHWRWUDQVGXFHGLYLGLQJFHOOV&RQVHTXHQWO\ the system was soon expanded to include lentiviruses, most commonly human LPPXQRGHĺFLHQF\YLUXV +,9 ZKLFKFDQLQIHFWFHOOVLQWKHDEVHQFHRIGLYLVLRQ 7KHXVHRI379IRUVHURORJLFDOHYDOXDWLRQLVZLGHO\UHSRUWHGDQGLQFUHDVLQJZLWK neutralization assays to detect neutralizing antibodies (nAbs) targeted towards WKH9(3KDYLQJEHHQGHYHORSHGIRUPDQ\YLUXVHVSURYLGLQJERWKVHQVLWLYHDQG VSHFLĺFUHVXOWVZKLFKFRUUHODWHZLWKOLYHYLUXVDVVD\V)RU5$%9DQGRWKHUO\VVD viruses, pseudotypes have previously been reported for different species, acting DVVXUURJDWHVIRUOLYHYLUXVWRHYDOXDWHOHYHOVRIFURVVQHXWUDOL]DWLRQDIIRUGHGE\ 5$%9YDFFLQHVDJDLQVW(XURSHDQEDWO\VVDYLUXV (%/9 DQG(4)7KHDVVD\ was further applied in a larger serosurveillance study in Africa, a developing FRXQWU\ ZKHUH UDELHV LV HQGHPLF GHWHFWLQJ UDELHV &96 LVRODWH DQWLERGLHV ZLWKLQĺHOGVHUXPVDPSOHVIURPYDFFLQDWHGGRJV%DVHGXSRQXQSXEOLVKHGGDWD further to this, pseudotypes have been generated to include at least one isolate from each lyssavirus species (5),QHDFKFDVHWKH3791$ZDVVKRZQWREHDEOH to distinguish between lyssavirus species and the results were shown to correlate ZLWKRULQVRPHFDVHVZHUHPRUHVHQVLWLYHWKDQWKHYDOLGDWHGOLYHYLUXV)$91 DVVD\ $GGLWLRQDOO\ LW ZDV GHPRQVWUDWHG KRZ VHURORJLFDO VWXGLHV YLD D 3791$ RQO\UHTXLUHDVPDOOYROXPHRIVHUXPIRUHDFKDVVD\ZKLFKLVtIROGORZHU WKDQDVVD\VZLWKOLYHYLUXV7KH\FDQDOVREHJHQHUDWHGWRHQFRGHRQHRIVHYHUDO UHSRUWHUJHQHV )LJ (6)%RWKRIWKHVHIDFWRUVHQKDQFHWKHLUVXLWDELOLW\WREH XVHGLQDZLGHUDQJHRIODERUDWRU\HQYLURQPHQWVDQGPLQLPL]HVFRVWV Pseudotype virus micro-neutralization assays Laboratory techniques in rabies Fifth edition Part 4. Demonstration of viral antibodies A )LUHĻ\ Renilla NEG luciferase luciferase Luminescence 8.DQG(GZDUG:ULJKW8QLYHUVLW\ % )OXRUHVFHQFH C Colourimetric )LJ([DPSOHVRISVHXGRW\SHYLUXVUHSRUWHUJHQHUHDGRXWV %\FRXUWHV\RI(PPD%HQWOH\8QLYHUVLW\RI:HVWPLQVWHU/RQGRQ RI6XVVH[%ULJKWRQ8. ,QIHFWLRQRIWDUJHWFHOOVZLWK379FDUU\LQJ $ OXPLQHVFHQFHUHSRUWHUJHQHVVXFKDVĺUHĻ\DQGRenillaOXFLIHUDVHDUHTXDQWLĺHGLQ UHODWLYHOLJKWXQLWV % ĻXRUHVFHQWUHSRUWJHQHVVXFKDV*)35)3DQGQXFOHDUORFDOL]HG*)3RU5)3FDQEHTXDQWLĺHGDVLQIHFWLRXV units; and (C) the colourimetric report gene lac=FDQEHGHWHFWHGXVLQJVHYHUDOGLIIHUHQWVXEVWUDWHV EURPRFKORURLQGRO\O¼' JDODFWRS\UDQRVLGH ;JDO 2QLWURSKHQ\O¼'JDODFWRS\UDQRVLGH 213* RUFKORURSKHQROUHG¼'JDODFWRS\UDQRVLGH &35* )RUHDFK JURXSRIJHQHVDQHJDWLYH 1(* UHVXOWLVDOVRVKRZQ Methods Generation of PTV stock 'D\ 6HHGKXPDQHPEU\RQLFNLGQH\ +(. 7FHOOVLQWRDZHOOFXOWXUH SODWH a[FHOOVZHOO RUFPFXOWXUHGLVK a[ cells) to be tFRQĻ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add the transfection reagent to the plasmid DNA and incubate for a IXUWKHUtPLQIUHTXHQWO\PL[LQJ7KHWUDQVIHFWLRQPL[LVWKHQDGGHG GURSZLVH WR WKH FHOOV ZKLOH VZLUOLQJ WKH FXOWXUH SODWHGLVK WR HYHQO\ GLVWULEXWH,QFXEDWHRYHUQLJKWLQ&&2LQFXEDWRU Pseudotype virus micro-neutralization assays Laboratory techniques in rabies Fifth edition Part 4. Demonstration of viral antibodies 'D\ $IWHU t K LQFXEDWLRQ DVSLUDWH RII WKH FHOO FXOWXUH PHGLXP DQG UHSODFHZLWKWKHHTXLYDOHQWYROXPH 'D\ +DUYHVW WKH FHOO FXOWXUH PHGLXP FRQWDLQLQJ WKH 379SDVV WKURXJK D }PFHOOXORVHEDVHGĺOWHUDQGVWRUHDW&5HSODFHZLWKWKHHTXL YDOHQWYROXPHRIPHGLDDQGFRQWLQXHLQFXEDWLRQ 'D\ +DUYHVWFHOOFXOWXUHPHGLXPFRQWDLQLQJWKH379DVEHIRUH&RPELQHWKH WZR379KDUYHVWV,GHDOO\VWRUHVWRFNVDWŗ&ZKHUHWKH\UHPDLQ VWDEOH IRU PDQ\ \HDUV RWKHUZLVH VWRUDJH DW ŗ & & DQG URRP WHPSHUDWXUHFDQEHXQGHUWDNHQIRUPRQWKVZHHNVRUGD\VUHVSHFWLYHO\ 7DEOH3ODVPLG'1$PL[IRUWUDQVIHFWLRQ Transfection reagent Culture dish Plasmid DNA PEI )XJHQH *DJSRO 5HSRUWHU Envelope 2SWL0(0 dHO ZHOO ŻJ ŻJ ŻJ Ż/ 8SWRŻ/ FP ŻJ ŻJ ŻJ Ż/ 8SWRŻ/ DNA, deoxyribonucleic acid; PEI, polyethyleneimine 7DEOH7UDQVIHFWLRQUHDJHQW Culture dish PEI )XJHQH PJP/3(, 2SWL0(0 5HDJHQW 2SWL0(0 ZHOO Ż/ Ż/ Ż/ Ż/ FP Ż/ Ż/ Ż/ Ż/ DNA, deoxyribonucleic acid; PEI, polyethyleneimine Titration of PTV stock 7KHWLWUHRI379FDQEHDVVHVVHGE\WLWUDWLQJRQWRDVXVFHSWLEOHWDUJHWFHOOOLQH W\SLFDOO\ %+. 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Demonstration of viral antibodies Colourimetric :KHQXVLQJ379LQFRUSRUDWLQJDODF=UHSRUWHUJHQHFHOOVDUHĺ[HGDVGHVFULEHG DERYHIRUWKHĻXRUHVFHQWUHSRUWHUV)ROORZLQJLQFXEDWLRQRIWKHFHOOVZLWK[3%6 IRUPLQDW&FHOOVDUHZDVKHGWZLFHZLWK[3%6EHIRUHEHLQJVWDLQHGE\ DGGLQJŻ/;JDO PJP/ GLOXWHGLQ;JDOEXIIHUIRUtPLQDW&3ODWHV VKRXOGEHZUDSSHGLQIRLOWRSURWHFWWKHFHOOVIURPOLJKW$IWHUVWDLQLQJFHOOVDUH ZDVKHGRQFHZLWK[3%6ZLWKŻ/DGGHGDQGOHIWRQHDFKZHOO7UDQVGXFHG cells, which present with blue stained nuclei, can then be viewed using a light PLFURVFRSH,QIHFWLRXVXQLWVFDQEHGHWHUPLQHGE\FRXQWLQJWKHQXPEHURIWUDQV GXFHGFHOOVZLWKLQHDFKZHOO Interpretation of results Neutralization titres of samples are recorded as the inhibitory concentration at ZKLFKRI379LQSXWLVQHXWUDOL]HG ,& )RUOXPLQHVFHQFHUHSRUWHUVWKLV LVZKHQWKH5/8YDOXHVUHDFKEDFNJURXQGOHYHOV VLPLODUWRWDUJHWFHOOVDORQHRU 379DORQHFRQWUROV IRUĻXRUHVFHQFHDQGFRORXULPHWULFUHSRUWHUVWKLVLVZKHUH QRĻXRUHVFHQWVWDLQHGFHOOVRUFRORXUFKDQJHLVREVHUYHG)RUZKLFKHYHUUHSRUWHU was used, the neutralization titres can be recorded as the concentration or reci procal dilution of the sample at which the IC HQGSRLQW LV DFKLHYHG :KHUH ,&HQGSRLQWWLWUHVYDU\E\PRUHWKDQRQHGRXEOLQJGLOXWLRQWKHDVVD\VKRXOG EHUHSHDWHGDQGWKHJHRPHWULFPHDQUHFRUGHG It is becoming increasingly common for pseudotype neutralization assays to EHPXOWLSOH[HGZKHUHVHSDUDWH379SUHSDUDWLRQEHDULQJGLIIHUHQW9(3RQWKHLU surface carry different reporter genes within their genomes (8–10)7KHXVHRIGLIIH UHQWUHSRUWHUJHQHVHQDEOHVWKHQHXWUDOL]DWLRQSRWHQF\RIHDFK379WREHDFFX UDWHO\GHWHUPLQHGLQWKHVDPHZHOOXVLQJWKHVDPSOHDOLTXRWRIVDPSOH+RZHYHU it is important to note that selection of reporter genes is critical and normally FRPSULVHV D FRPELQDWLRQ RI OXPLQHVFHQW ĻXRUHVFHQW DQG FRORXULPHWULF JHQHV 7KHXVHRIVHFUHWHGUHSRUWHUSURWHLQVLVKLJKO\GLVFRXUDJHGIRUPRQRRUPXOWLSOH[ 379DVVD\VDVWKHUHSRUWHUZLOOEHH[SUHVVHGLQWRWKHFHOOFXOWXUHPHGLXPGXULQJ 379SURGXFWLRQDQGWKHUHIRUHEHSUHVHQWZLWKLQVWRFNVRIKDUYHVWHG3797KLVZLOO OHDGWRWKHIDOVHtSRVLWLYHGHWHFWLRQRIUHSRUWHUDFWLYLW\LQGRZQVWUHDPDVVD\V Pseudotype virus micro-neutralization assays Laboratory techniques in rabies Fifth edition Part 4. 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Demonstration of viral antibodies Annex s '0(0tKLJKJOXFRVH PJ/JOXFRVH /JOXWDPLQHVRGLXPS\UXYDWHDQG VRGLXPELFDUERQDWH/LTXLGVWHULOHĺOWHUHGDQGVXLWDEOHIRUFHOOFXOWXUH6XSSOH PHQWHGZLWKIHWDOERYLQHVHUXPXQLWVSHQLFLOOLQP/DQG}JVWUHS WRP\FLQP/ s 2SWL0(0tUHGXFHGVHUXPPHGLXP*OXWD0$;OLTXLGVXSSOHPHQWDQGFRQWDLQV SKHQROUHG • Transfection reagents: )X*(1(WUDQVIHFWLRQUHDJHQW 3URPHJD( 3(,WUDQVIHFWLRQUHDJHQW EUDQFKHG 6LJPD LVPDGHXSDWPJP/ LQGLVWLOOHGZDWHUG+2S+DGMXVWHGWRZLWKGLOXWH+&O>K\GURFKORULFDFLG@ VWHULOL]HGXVLQJDPĺOWHUDQGVWRUHGDWŗ&XQWLOQHHGHG,WFDQWKHQ EHNHSWDW & IRUPRQWK s %ULJKW*ORDVVD\V\VWHP 3URPHJD( s ;*$/VXEVWUDWHEXIIHUtPPROGHR[\FKROLFDFLGPPROSRWDVVLXPIHUURF\D QLGHPPROPDJQHVLXPFKORULGHDQG YY 13;JDOLVGLVVROYHG LQGLPHWK\OIRUPDPLGH s ¼JDODFWRVLGDVHVXEVWUDWHV ;JDO 6LJPD% 213* 6LJPD1 &35* 6LJPD Pseudotype virus micro-neutralization assays Laboratory techniques in rabies Fifth edition Part 4. Demonstration of viral antibodies Chapter 24 #SKMPĚKĂGFĝTORGSEGNEGKNJKDKěKONMKEROěGSě for the determination of rabies virus neutralizing antibodies Introduction :+2 UHFRPPHQGV WKDW DQ\RQH ZKR LV DW ULVN IRU H[SRVXUH WR UDELHV YLUXV 5$%9 EHFDXVHRIWKHLUUHVLGHQFHRURFFXSDWLRQLQFOXGLQJODERUDWRU\ZRUNHUV GHDOLQJ ZLWK 5$%9 DQG RWKHU O\VVDYLUXVHV YHWHULQDULDQV DQG DQLPDO KDQGOHUV VKRXOGUHFHLYHSUHH[SRVXUHYDFFLQDWLRQ(1)6XFKSHUVRQVZRUNLQJZLWKOLYHYLUXV in diagnostic laboratories, research laboratories and vaccine production laborato ULHVHWFZKRDUHDWFRQWLQXRXVULVNRIH[SRVXUHWRUDELHVVKRXOGKDYHWKHHYDOXD WLRQRIYLUXVQHXWUDOL]LQJDQWLERGLHV 91$ SHUIRUPHGSHULRGLFDOO\HYHU\PRQWKV IRURWKHUSURIHVVLRQV YHWHULQDULDQVDQLPDOKDQGOHUVZLOGOLIHRIĺFHUVHWF ZRUNLQJ LQDUHDVHQGHPLFIRUUDELHVLWVKRXOGEHHYHU\\HDUV$OOSHUVRQVVKRXOGKDYHD ERRVWHUGRVHZKHQWKH91$WLWUHDJDLQVW5$%9IDOOVEHORZ,8P/(1, 2) 9DULRXVYLUXVQHXWUDOL]DWLRQWHVWVDUHPRVWFRPPRQO\XVHGIRUWKHGHWHFWLRQ RI91$,Q :HEVWHUDQG'DZVRQGHYHORSHGWKHPRXVHQHXWUDOL]DWLRQWHVW 017 IRU GHWHFWLRQ RI 91$ ZLWK WKH LQRFXODWLRQ RI UHVLGXDO 5$%9 LQ PLFH (3) :LWK}WKHDGDSWDWLRQRIWKHFKDOOHQJHYLUXVVWDQGDUG &96 LQFHOOFXOWXUHDQLQYLWUR WHFKQLTXHZDVGHYHORSHGDVDUHSODFHPHQWRI017WKHUDSLGĻXRUHVFHQWIRFXV LQKLELWLRQWHVW 5)),7 SHUIRUPHGRQZHOOWLVVXHFXOWXUHFKDPEHUVOLGHV7KHXVH RIZHOOVOLGHVOLPLWVWKHQXPEHURIVHUXPVDPSOHVWHVWHGDQGFRPSOLFDWHVDXWR mation (4)=DODQDQGFROOHDJXHV(5)GHYHORSHGDPLFURWHVWIRUWKHTXDQWLĺFDWLRQRI UDELHV91$EDVHGRQ5)),7XVLQJ ZHOOPLFURSODWHVWKHĻXRUHVFHQFHLQKLELWLRQ PLFURWHVW ),07 7KH),07ZDVFRQVLGHUHGPRUHUDSLGDQGHFRQRPLFDOWKDQWKH 5)),7 %DVHGRQWKH5)),7DQGWKH),07GXULQJ DVLPSOLĺHGĻXRUHVFHQFHLQKL ELWLRQPLFURWHVW 6),07 ZDVVWDQGDUGL]HGDQGLPSOHPHQWHGDWWKHODERUDWRU\IRU UDELHVGLDJQRVLVRIWKH3DVWHXU,QVWLWXWHLQ6À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wildlife is an important tool for epidemiological assessments that may underpin WKH GHYHORSPHQW RI GLVHDVH SUHYHQWLRQ DQG FRQWURO VWUDWHJLHV 6HYHUDO VWXGLHV XVLQJ6),07KDYHGHWHFWHGORZWLWUHVRIUDELHV91$LQZLOGFDUQLYRUHVDQGRWKHUV ZLOGOLIHLQ%UD]LO(8–11) 2HLOKHjDCkTNQDRBDMBDHMGHAHSHNMLHBQNSDRS Laboratory techniques in rabies Fifth edition Part 4. 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Demonstration of viral antibodies References :+2([SHUW&RQVXOWDWLRQRQ5DELHVWKLUGUHSRUW*HQHYD :RUOG+HDOWK2UJD QL]DWLRQ :+27HFKQLFDO5HSRUW6HULHV1RKWWS DSSVZKRLQWLULV ELWVWUHDPKDQGOH BHQJSGI DFFHVVHG0D\ :+2*XLGHIRUUDELHVSUHDQGSRVWH[SRVXUHSURSK\OD[LVLQKXPDQV*HQHYD :RUOG +HDOWK 2UJDQL]DWLRQ KWWS ZZZZKRLQWUDELHV3(3B3URSK\OD[LVB JXLGHOLQHBBBSGIDFFHVVHG0D\ :HEVWHU/7'DZVRQ-/(DUO\GLDJQRVLVRIUDELHVE\PRXVHLQRFXODWLRQ0HDVXUH PHQWRIKXPRUDOLPPXQLW\WRUDELHVE\PRXVHSURWHFWLRQWHVW3URF6RF([S%LRO 0HG t 6PLWK- 2HLOKHjDCkTNQDRBDMBDHMGHAHSHNMLHBQNSDRS Laboratory techniques in rabies Fifth edition Part 4. 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Demonstration of viral antibodies Chapter 25 The immunoperoxidase inhibition assay Introduction The immunoperoxidase inhibition assay (IIA) is an alternative test for the detec WLRQRIUDELHVYLUXV 5$%9 DQWLERGLHVLQVHUD7KH,,$LVDEORFNLQJDVVD\EDVHGRQ WKHUHDFWLRQEHWZHHQWHVWVHUDDQG5$%9DQWLJHQVLQLQIHFWHGFHOOV3RVLWLYHWHVW VHUDZLOOLQKLELWOLQNDJHRIDQDQWL5$%9SRO\FORQDOVHUXP SUHSDUHGLQUDEELWV ZKLFKZLOOWKHQEHUHYHDOHGE\WKHDGGLWLRQRIDQDQWLUDEELW,J*SHUR[LGDVHFRQMX JDWH7KHWHVWLVORZLQFRVWDQGGRHVQRWUHTXLUHVSHFLĺFHTXLSPHQWVXFKDVD ĻXRUHVFHQFHPLFURVFRSHRQO\EDVLFFHOOFXOWXUHIDFLOLWLHV7KHUHVXOWVDUHHDVLO\ read in an ordinary light microscope and interpretation is straightforward, with UHDGLQJEDVHGRQDQqDOORUQRWKLQJrSULQFLSOH:KHQFRPSDUHGWRWKHqVLPSOLĺHG ĻXRUHVFHQFHLQKLELWLRQPLFURWHVWr(1) with human sera, it revealed a strong positive correlation (2)$VWKH,,$LVEDVHGRQDEORFNLQJUHDFWLRQLWPLJKWDOVREHSRWHQ WLDOO\XVHIXOIRUGHWHFWLQJ5$%9DQWLERGLHVLQDQ\VSHFLHVZLWKRXWVSHFLĺFFRQMX JDWHV7KLVDVVD\PD\EHDGYDQWDJHRXVRYHURWKHUVLPLODUPHWKRGVGHVLJQHGWR GHWHFW5$%9VSHFLĺFELQGLQJDQWLERGLHVEHFDXVHLWFDQEHHDVLO\LQWURGXFHGLQWR PRVWODERUDWRULHVSURYLGHGEDVLFFHOOFXOWXUHIDFLOLWLHVDUHDYDLODEOH Advantages of the IIA The IIA is advantageous for the following reasons: • does not utilize competing monoclonal antibodies; s GRHVQRWUHTXLUHSUHSDUDWLRQRIODUJHYROXPHVRIYLUXVQRUSXULĺFDWLRQRIYLUDO proteins for antigen preparation; • does not require expensive equipment for antigen preparation or ELISA plate readers; s GRHVQRWUHTXLUHDĻXRUHVFHQFHPLFURVFRSH UHDGLQJVFDQEHSHUIRUPHGZLWK WKHDLGRIDUHJXODUOLJKWPLFURVFRSHZLWKD[RU[REMHFWLYHPDJQLĺFDWLRQ s WKHSRO\FORQDOUDELHVDQWLVHUXPUHTXLUHGFDQEHSUHSDUHGORFDOO\ DQG • the test can be potentially useful for the detection of antibodies in different DQLPDOVSHFLHV Immunoperoxidase inhibition assay Laboratory techniques in rabies Fifth edition Part 4. Demonstration of viral antibodies Protocol 1.1 Cells s )RU WKH ,,$ FXOWLYDWH WKH FKLFNHQ HPEU\R UHODWHG &(5 FHOO OLQHDJH (3) in (DJOHpV0LQLPXP(VVHQWLDO0HGLXP (0(0 VXSSOHPHQWHGZLWKIHWDO ERYLQHVHUXP )%6 DQGDQWLELRWLFV ,8P/SHQLFLOOLQJP/VWUHS WRP\FLQ VSOLWWLQJFHOOVRQFHZHHNO\ 1.2. Viruses s )RUWKH,,$SURSDJDWH5$%9VWUDLQ3DVWHXUYLUXV 39 LQ&(5FHOOV SUHSDUHG DVLQ s )RUSURGXFWLRQRI5$%9DQWLVHUXPLQUDEELWVXVHWKHFKDOOHQJHYLUXVVWDQ GDUG &96 DIWHUYDFFLQDWLRQ$PSOLI\&96LQPLFHDJHGtZHHNVIROORZLQJ standard procedures (see Chapter 8RQ9LUXVLVRODWLRQLQDQLPDOV WKHPRXVH inoculation test) or prepare the virus in cell culture as described by Caporale DQGFROOHDJXHV}(4) 1.3. Preparation of polyclonal rabbit anti-rabies serum s 9DFFLQDWHUDEELWVLQWUDPXVFXODUO\ZLWKWKUHHZHHNO\GRVHVRIDFRPPHU FLDOO\DYDLODEOHUDELHVYDFFLQH s 7ZHQW\RQHGD\VDIWHUWKHODVWLPPXQL]DWLRQFKDOOHQJHWKHUDEELWVZLWK OHWKDOGRVHV /' RIVWUDLQ&96 s &ROOHFWVHUXPVDPSOHVIURPHDFKDQLPDOZHHNVDIWHUFKDOOHQJH • Titrate serum samples from each animal with a usual antibody detection DVVD\DQGXVHDVDVHFRQGDU\DQWLERG\LQWKH,,$7KHDSSURSULDWHGLOXWLRQRI WKHSRO\FORQDOUDEELWDQWLUDELHVVHUXPWREHXVHGLQWKH,,$LVGHWHUPLQHGE\ WHVWLQJVHULDOIROGGLOXWLRQVRIWKHVHUXPLQWKHWHVWZLWKWKHSRVLWLYHUHIH UHQFHVHUXP GLOXWHGWR,8P/ DQGWKHDQWLERG\QHJDWLYHFRQWUROVHUXP DVDVRXUFHRIqEORFNLQJrDQWLERGLHV7KHKLJKHVWVHUXPGLOXWLRQFDSDEOH RI ELQGLQJ WR DQWLJHQFRQWDLQLQJ FHOOV ZLWK D FOHDU SRVLWLYH UHDFWLRQ DIWHU VWDLQLQJDQGDPLQLPDOEDFNJURXQGVWDLQLQJVKRXOGEHVHOHFWHGIRUXVHLQ VXEVHTXHQWWHVWV s 0DNH SRO\FORQDO UDEELW DQWLUDELHV VHUXP LQ GLOXWLRQ EXIIHU as described LQb • Perform the animal handling procedures according to institutional guidelines for animal welfare for experimentation on animals (KWWS ZZZRLHLQWLQGH[ SKS"LG / KWPĺOH WLWUHBKWP 1.4. 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Preparation of serum samples s ,QDFWLYDWHWKHVHUDWHVWDW&IRUPLQ s 'LOXWHDQLQWHUQDWLRQDOUHIHUHQFHVHUXPWRDĺQDOFRQFHQWUDWLRQRI,QWHU QDWLRQDO8QLWVRI5$%9QHXWUDOL]LQJDQWLERGLHVSHUP/ ,8P/ DQGLQFOXGHLQ DOOH[SHULPHQWVDVSRVLWLYHFRQWUROVHUXP s ,ISRVVLEOH DIWHU+XPDQ6XEMHFWV5HYLHZDQGIROORZLQJDOOELRVDIHW\SUHFDX tions for the use of human blood products), use serum collected from a person not previously vaccinated against rabies as a negative control, inac WLYDWHDVGHVFULEHGDERYHDQGGLOXWH IRUXVHLQDOOH[SHULPHQWV Considerations 0DNHGLOXWLRQVRIWHVWVHUDDVZHOODVSRVLWLYHDQGQHJDWLYHFRQWUROVHUDLQGLOXWLRQ buffer (as described in 2.1 1.6. 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Demonstration of viral antibodies s $GGŻ/ZHOORI3%6 s 5HDGWKHSODWHVRQDVWDQGDUGLQYHUWHGPLFURVFRSHZLWKD[PDJQLĺFDWLRQ REMHFWLYH Considerations – 0DNHDOOGLOXWLRQVRIWHVWVHUDSRO\FORQDOUDEELWDQWLUDELHVVHUXPDQGWKH DQWLUDEELW ,J* SHUR[LGDVH FRQMXJDWH DQG FRQWUROV LQ GLOXWLRQ EXIIHU as described in 2.1 – 8VH DQWLERG\SRVLWLYH VHUD ZKHQ LW LV FDSDEOH RI LQKLELWLQJ RI WKH ELQGLQJRIWKHSRO\FORQDOUDEELWDQWLUDELHVVHUXPWKXVSUHFOXGLQJELQGLQJ RIWKHFRQMXJDWHJLYLQJULVHWRXQVWDLQHGZHOOV – 8VHDQWLERG\QHJDWLYHVHUDZKHQLWLVLQFDSDEOHRILQKLELWLQJWKHELQGLQJRI WKHSRO\FORQDOUDEELWDQWLUDELHVVHUXPJLYLQJULVHWRVWDLQHGZHOOV – 7KHSHUR[LGDVHFRQMXJDWHUHDFWVZLWKK\GURJHQSHUR[LGHLQWKHSUHVHQFH RI WKH VXEVWUDWH $(& ZKLFK SUHFLSLWDWHV RQ WKH DQWLJHQFRQWDLQLQJ FHOOV DOORZLQJYLVXDOL]DWLRQRIWKHFKDUDFWHULVWLFFDUPLQHUHGF\WRSODVPLFVWDLQLQJ – Calculate the antibody titre of the test sera on the basis of the last dilution FDSDEOHRIEORFNLQJRIWKHVSHFLĺFVWDLQLQJ – &RQVLGHUWKHWHVWYDOLGLIWKHUHIHUHQFHVHUXP GLOXWHGWR,8P/ LVDEOH WREORFNRIWKH$(&FRORXUUHDFWLRQE\FRPSHWLQJIRUDQWLJHQZLWKWKH SRO\FORQDOUDELHVDQWLVHUXP/LNHZLVHSRO\FORQDO5$%9DQWLVHUXPELQGLQJ VKRXOGQRWEHLQKLELWHGE\WKHQHJDWLYHFRQWUROVHUXP GLOXWHG VKRZQ E\DFRORXUHGUHDFWLRQLQWKHZHOOV – ([SUHVVWKH,,$UHVXOWVLQHTXLYDOHQWXQLWVSHUP/RIVHUXP (8P/ E\FDOLEUD WLRQ DJDLQVW WKH LQWHUQDWLRQDO UHIHUHQFH VHUXP $QWLERG\ WLWUHV ł} } (8 mL should be considered positive for antibodies, whereas antibody titres }}(8P/VKRXOGEHFRQVLGHUHGQHJDWLYH(6) Description of solutions 2.1 Dilution buffer J1D&O J1DHPO J1DHPO7ZHHQ+2TVS/ )L[DWLRQEXIIHU SDUDIRUPDOGHK\GHLQ3%6 J1D&OJ1DHPOJ1DHPO, HO TVS/S+ 2.3 Wash buffer 7ZHHQLQ3%6S+ Immunoperoxidase inhibition assay Laboratory techniques in rabies Fifth edition Part 4. Demonstration of viral antibodies References )DYRUHWWR65&DUULHUL0/7LQR06=DQHWWL&53HUHLUD2$&6LPSOLĺHGĻXRUHV FHQWLQKLELWLRQPLFURWHVWIRUWKHWLWUDWLRQRIUDELHVQHXWUDOL]LQJDQWLERGLHV5HY,QVW 0HG7URS6DR3DXOR t %DWLVWD+%&5/LPD)(60DOHWLFK'6LOYD$&59LFHQWLQL).95RHKH/5HW DO,PPXQRSHUR[LGDVHLQKLELWLRQDVVD\ ,,$ IRUUDELHVDQWLERG\GHWHFWLRQ-9LURO 0HWK t &DUGRVR 7& 4XHLUR] GD 6LOYD /+ $OEDV $ )HUUHLUD +/ 3HUUL 6+95DELHV neutralizing antibody detection by indirect immunoperoxidase serum neutraliza WLRQ DVVD\ SHUIRUPHG RQ FKLFNHQ HPEU\R UHODWHG FHOO OLQH 0HP ,QVW 2VZDOGR &UX] tGRL 6 &DSRUDOH*006LOYD$&53HL[RWR=03&KDYHV/%&DUULHUL09DVVÀR5& )LUVWSURGXFWLRQRIĻXRUHVFHQWDQWLULERQXFOHRSURWHLQVFRQMXJDWHIRUGLDJQRVLVRI UDELHVLQ%UD]LO-&OLQ/DE$QDO t +DUORZ(/DQH'$QWLERGLHV DODERUDWRU\PDQXDOQGHGLWLRQ1HZ Immunoperoxidase inhibition assay Laboratory techniques in rabies Fifth edition Part 4. Demonstration of viral antibodies Annex. Reagents, equipment and biological materials required for the IIA Reagents s(DJOHpV0LQLPXP(VVHQWLDO0HGLXP (0(0 sIHWDOERYLQHVHUXP )%6 • trypsin sDQWLELRWLFV ,8P/SHQLFLOOLQJP/VWUHSWRP\FLQ sVXEVWUDWHDPLQR HWK\OFDUED]ROH $(& sSKRVSKDWHEXIIHUHGVDOLQH 3%6 sDQWLUDEELW,J*SHUR[LGDVHFRQMXJDWH Equipment s%LRORJLFDO6DIHW\&DELQHW • standard inverted microscope • CO incubator • freezer Animals sPLFHDJHGtZHHNV • rabbits Biological materials sFKLFNHQHPEU\RUHODWHG &(5 FHOOOLQHDJH s3DVWHXUYLUXV 39 sFKDOOHQJHYLUXVVWDQGDUG &96 • rabies vaccine • international reference serum • negative control serum Immunoperoxidase inhibition assay Laboratory techniques in rabies Fifth edition Part 4. Demonstration of viral antibodies Chapter 26 Demonstration of rabies virus antibodies by the counter immunoelectrophoresis test Introduction 7LWUDWLRQRIDQWLERGLHVDOORZVWKHLPPXQLW\RILQGLYLGXDOVZKRUHFHLYHDQWLUD ELHVSUHRUSRVWH[SRVXUHSURSK\OD[LV 3(3 WREHTXDQWLĺHG$WLWUHRI,8P/ in the serum neutralization test is considered the minimum adequate value indica tive of an individual response after prophylaxis (1)6XFKWHVWVDUHXVHGWRTXDQWLI\ YLUXVQHXWUDOL]LQJDQWLERGLHV 91$ DJDLQVWUDELHVYLUXV 5$%9 (1–3)7KHPRXVH QHXWUDOL]DWLRQWHVW 017 WKHĺUVWWREHGHYHORSHG(4, 5), was long considered as the reference test; however, it is costly due to animal use (6) and also raises ethical DQGDQLPDOZHOIDUHLVVXHV7KHUDSLGĻXRUHVFHQWIRFXVLQKLELWLRQWHVW 5)),7 RULJL nally developed by Smith and colleagues (7)DQG=DODQDQGFROOHDJXHV(8), was the WHVWXVHGWRDGDSWWKHWHFKQLTXHWRFHOOFXOWXUH7KH5)),7UHYHDOVWKHSUHVHQFHRU DEVHQFHRIYLUDOLQIHFWLRQLQFHOOFXOWXUHDIWHUaKXVLQJWKHĻXRUHVFHQWDQWLERG\ technique (9)7KH5)),7 VHHDOVRChapter 19 WRJHWKHUZLWKWKH)$91 ĻRUHVFHQW antibody virus neutralization) test (see also Chapter 20), are now considered the gold standard techniques (7, 9)GXHWRWKHLUJRRGFRUUHODWLRQZLWKWKH017 VHHDOVR Chapter 22 and references 2, 10–12 Counter immunoelectrophoresis (CIE) is another technique for the titration RI5$%9VSHFLĺFDQWLERGLHV7KH&,(GHWHFWVDQWLERGLHVRIWKH,J*FODVVYLUDO DQWLJO\FRSURWHLQ WKH PDLQ LPPXQRJOREXOLQ LQYROYHG LQ UHVSRQVH DJDLQVW WKH YLUXV)RUWKLVUHDVRQWKH&,(FDQGHWHUPLQHWKHQHXWUDOL]LQJSRWHQWLDORIDQDO\VHG sera (13, 14) 7KH &,( LV SHUIRUPHG RQ DJDURVH JHO VOLGHV DQG LV EDVHG RQ WKH reaction of the antibodies present in serial dilutions of the test serum against a FRQVWDQWFRQFHQWUDWLRQRIYLUDODQWLJHQ$IWHUDĺUVWHOHFWURSKRUHWLFUXQWKHK\SH ULPPXQHLQGLFDWRUVHUXPLVORFDWHGWRDOORZIRUGLIIXVLRQLQWKHRSSRVLWHGLUHFWLRQ $SUHFLSLWDWLRQOLQHIRUPVDWDQ\VHUXPGLOXWLRQZKHUHQRDQWLERGLHVDUHSUHVHQW 7KH}WHFKQLTXHLVVLPSOHORZFRVWDQGSURYLGHVUHVXOWVLQDVKRUWSHULRGRIWLPH tK DQGWKHUHIRUHLVRIXWLOLW\IRUFOLQLFDOSUDFWLFH(13, 15–18) 7KHFRPSDULVRQRI5$%9$EWLWUHVGHWHFWHGE\DPRGLĺHG&,( 0&,( DQG017 show good correlation between these techniques (13))XUWKHUZKHQFRPSDUHG ZLWK WKH VWDQGDUG 017 DQG 5)),7 WHFKQLTXHV 0&,( KDG JRRG VHQVLWLYLW\ DQG VSHFLĺFLW\IRUHVWLPDWLQJDQWLERGLHV(19, 20),QDGGLWLRQWKH0&,(DQG017ZHUH not obviously different regarding the titre of detected antibodies (21) 6HYHUDO studies using this technique have been published (22, 23)0RUHRYHUWKH 0&,( was shown to be sensitive enough to detect the minimum acceptable level of $E ,8P/ (18) Counter immunoelectrophoresis test Laboratory techniques in rabies Fifth edition Part 4. Demonstration of viral antibodies Protocol The CIE is performed according to the general procedure described by Diaz DQG0\HUV 13 &HQWULIXJHWKHVHUDRUSODVPDWREHWHVWHG 76 DWgIRUPLQ 7UDQVIHUWKHVXSHUQDWDQWLQWRDVWHULOHWXEHLQDFWLYDWHGDW&IRUPLQ LQDZDWHUEDWK 6WRUHDWŗ&RUORZHU $WWKHWLPHRIWLWUDWLRQWKDZWKHVHUDDQGSUHSDUHDVHULDOGLOXWLRQT ZLWK3%6S+VWDUWLQJDW QHDW WRZLWKŻ/YROXPHVSHUWXEH ĺQDOGLOXWLRQVWR $GGŻ/RIWKHVWDQGDUGL]HG$JWRSUHGLOXWLRQZLWKWZLFHWKHZRUNLQJ FRQFHQWUDWLRQ IRUH[DPSOHZRUNLQJGLOXWLRQ SUHGLOXWHGWR LQ DOO76WXEHVDQGŻ/LQWKHQHJDWLYHFRQWUROWXEHFRQWDLQLQJŻ/RI negative serum (NS), so that the Ag concentration falls to the ideal (half) DIWHUPL[LQJ ,QFXEDWHWKHVHUXPt$JPL[WXUHDW&IRUPLQ 3HUIRUPWKHHOHFWURSKRUHWLFUXQRQ DJDURVHJHOVOLGHVPHOWHGLQD ZDWHUEDWKRUPLFURZDYHRYHQDW & 7RSURGXFHWKHVOLGHVSRXUWKHYROXPHRIP/RQWRWKH´PPDQG P/IRUWKH´PPFRQWUROVOLGHVRQDOHYHOVXUIDFH /HWWKHJHOVOLGHVVROLGLI\DQGNHHSWKHPUHIULJHUDWHGLQDFORVHGFKDPEHU IRUPLQ 0DNHWZRVHWVRIĺYHZHOOVRQWKHJHOXVLQJRQHFDUGERDUGWHPSODWHXQGHU HDFKJODVVVOLGHRQHPPVHULHVIRUWKHVHUXPt$JPL[WXUHDQGRQHPP IRUWKHK\SHULPPXQHLQGLFDWRUVHUXP ,6 DWDGLVWDQFHRItPPIURP HDFKRWKHU ,Q WKH FRQWURO VOLGH PDNH WZR ZHOOV RI PP DQG WZR ZHOOV RI PP )LJ} /HDYHWKHFXWJHOSOXJVLQWKHZHOOVE\LPPHGLDWHO\ZLWKGUDZLQJWKHPP ZHOOVDQGSODFHWKHVOLGHVLQWKHHOHFWURSKRUHVLVFKDPEHUĺOOHGZLWKERUDWH EXIIHUS+ 3RVLWLRQWKHPPZHOOVRQWKHDQRGHVLGH SRVLWLYH DQGWKHPPZHOOV RQWKHFDWKRGHVLGH QHJDWLYH 'LVWULEXWHWKHVHUXPt$JPL[WXUHLQWKHPPZHOOVFRQWDLQLQJ76DQGWKH QHJDWLYHFRQWUROVOLGH &DUU\RXWWKHĺUVWUXQZLWKDFRQVWDQWFXUUHQWRIP$SHUVOLGHIRUPLQ NHHSLQJWKHFRQQHFWLQJZLFNVPRLVWHQHGFRQVWDQWO\ $IWHU WKLV SHULRG GLVWULEXWH WKH ,6 LQ WKH PP ZHOOV DQG SURFHHG ZLWK DQRWKHUHOHFWURSKRUHVLVUXQIRUPLQ 7KHHOHFWURSKRUHWLFUXQLVSHUIRUPHGXVLQJHTXLSPHQWVXFKDVDQHOHF trophoresis power supply, voltmeter, electrophoresis chamber, biosafety FDELQHWFODVV,,$UHIULJHUDWHGFHQWULIXJHWKDWUHDFKHVg, and water EDWK DW & DQG & VHH )LJV t 7KH ZLFNV FRQQHFWLQJ WKH VOLGHVDQGEXIIHUFDQEHPDGHZLWKĺOWHUSDSHURUYLVFRVHIDEULF )LJ Counter immunoelectrophoresis test Laboratory techniques in rabies Fifth edition Part 4. 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Demonstration of viral antibodies References :+2([SHUW&RQVXOWDWLRQRQ5DELHVWKLUGUHSRUW*HQHYD :RUOG+HDOWK2UJD QL]DWLRQ :+27HFKQLFDO5HSRUW6HULHV1RKWWS DSSVZKRLQWLULV ELWVWUHDPKDQGOH HQJSGIDFFHVVHG0D\ 6PLWK-65DELHVVHURORJ\,Q %DHU*0HGLWRU7KHQDWXUDOKLVWRU\RIUDELHV QGHGLWLRQ%RVWRQ 0$ &5&3UHVV t &OLTXHW)$XEHUW06DJQH,'HYHORSPHQWRIĻXRUHVFHQWDQWLERG\YLUXVQHXWUD OL]DWLRQ WHVW )$91 WHVW IRU WKH TXDQWLWDWLRQ RI UDELHVQHXWUDOLVLQJ DQWLERG\ - ,PPXQRO0HWKRGV tGRL 6 :HEVWHU /7 'DZVRQ -5 (DUO\ GLDJQRVLV RI UDELHV E\ PRXVH LQRFXODWLRQ 0HDVXUHPHQWRIKXPRUDOLPPXQLW\WRUDELHVE\PRXVHSURWHFWLRQWHVW3URF6RF ([S%LRO0HG $WDQDVLX34XDQWLWDWLYHDVVD\DQGSRWHQF\WHVWRIUDELHVVHUXPDQGLPPX QRJOREXOLQ,Q 0HVOLQ);.DSODQ00.RSURZVNL+HGLWRUV/DERUDWRU\WHFK QLTXHVLQUDELHVWKHGLWLRQ*HQHYD :RUOG+HDOWK2UJDQL]DWLRQ t 5L]]R/)>+XPDQDQWLUDELHVLPPXQRSURK\OD[LVZLWKODWHQWPRXVHEUDLQYDFFLQH XVLQJUHGXFHGYDFFLQDWLRQVFKHGXOHV@5HY8QLY6DQ&DUORV t LQ 6SDQLVK 6PLWK-6 Counter immunoelectrophoresis test Laboratory techniques in rabies Fifth edition Part 4. Demonstration of viral antibodies 'LD] $0 $ULVSH ( %UXQHO & &DYDQGROL & 'HOOHSLDQH 1 0LUDQGD $ >7KH FRXQWHULPPXQRHOHFWURSKRUHVLV WHFKQLTXH IRU WKH GHWHUPLQDWLRQ RI DQWLUDELHV DQWLERGLHV@%RO2ĺF6DQLW3DQDPHU t LQ6SDQLVK 'LD]$0$OEDV$9DOHQWLQL(-*3HUGRPR*>(YDOXDWLRQRIWKHTXDOLW\RIWKH reagents used in the counterimmunoelectrophoresis technique for the determi QDWLRQRIDQWLUDELHVDQWLERGLHV@5HY,QVW0HG7URS6ÀR3DXOR t LQ 6SDQLVK &KDXKDQ$6RRG'.6DKD60.DSRRU0$JJDUZDO5.6D[HQD617KHTXDQ WLWDWLRQRIUDELHVVSHFLĺFDQWLERGLHV,0RGLĺHGFRXQWHULPPXQRHOHFWURSKRUHVLV $ UDSLG DQG VHQVLWLYH PHWKRG %LRORJLFDOV t GRL 6LOYD /+4 %LVVRWR &( &DUYDOKR & &DUGRVR 7& 3LQKHLUR '0 3HUUL 6+9 Comparison between the counterimmunoelectrophoresis test and mouse neutra OL]DWLRQWHVWIRUWKHGHWHFWLRQRIDQWLERGLHVDJDLQVWUDELHVYLUXVLQGRJVHUD0HP ,QVW2VZDOGR&UX] tGRL 6 'LD]$05DELHVQHXWUDOL]LQJDQWLERGLHVGHWHUPLQDWLRQE\WKHPRGLĺHGFRXQ WHULPPXQRHOHFWURSKRUHVLV WHVW DQG UDSLG ĻXRUHVFHQW IRFXV LQKLELWLRQ WHVW =EO %DNW0LNURE+\J $ t &KDXKDQ$6RRG'.6DKD60.DSRRU0$JJDUZDO5.6D[HQD617KH TXDQWLWDWLRQRIUDELHVVSHFLĺFDQWLERGLHV,,,$FRPSDUDWLYHHYDOXDWLRQRIPRGLĺHG counter immunoelectrophoresis, haemagglutination inhibition and serum neutra OL]DWLRQ WLWUHV RI KXPDQ VHUD %LRORJLFDOV t GRL $OEDV$3DUGR3(5LQDOGL3/)7DUXPRWR0+>(YDOXDWLRQRIWKHFRXQWHULP PXQRHOHFWURSKRUHVLV PHWKRG WR WLWUDWH ERYLQH VHUD YDFFLQDWHG DJDLQVW UDELHV@ 5HY0HG9HW=RRW81(63 t LQ3RUWXJXHVH 2OLYHLUD $1 $QGUDGH 0&5 6LOYD 090RXUD :& &RQWUHLUDV (& ,PPXQH UHVSRQVH LQ FDWWOH YDFFLQDWHG DJDLQVW UDELHV 0HP ,QVW 2VZDOGR &UX] t 4XHLUR]/+0RUHLUD:&0RXUD:&6LOYD09'HPRQVWUDWLRQRIYLUDODQWLER GLHVE\WKHFRXQWHULPPXQRHOHFWURSKRUHVLVWHVW,Q 5XSSUHFKW&1DJDUDMDQ7 HGLWRUV&XUUHQWODERUDWRU\WHFKQLTXHVLQUDELHVGLDJQRVLVUHVHDUFKDQGSUHYHQ WLRQYRO1HZ Counter immunoelectrophoresis test Laboratory techniques in rabies Fifth edition Part 4. Demonstration of viral antibodies #NNGW%OMPOSKěKONOHMGFKCDTėGRSCNF solutions for the CIE test Phosphate buffered saline pH 7.2 • sodium phosphate dibasic (NaHPO J s VRGLXPFKORULGH 1D&O J • potassium phosphate monobasic (KHPO J s SXULĺHGZDWHU FOLQLFDOODERUDWRU\UHDJHQWZDWHU&/5: TV P/ $GMXVWS+WRDQGVWHULOL]HDW&IRUPLQ Agarose gel 0.9% s DJDURVHW\SH,,PHGLXP((2 J s YHURQDOEXIIHUTV P/ 3UHSDUHWKHVXVSHQVLRQLQDZDWHUEDWKEHORZ &'LVWULEXWHLQDOLTXRWVRI P/DQGNHHSDWt&XQWLOXVH 0.05 M veronal buffer pH 8.2 s GLHWK\OEDUELWXUDWHDFLGVRGLXP &HNNaO J s &/5:TV P/ $GMXVWS+WR Borate buffer pH 9.1 • boric acid (H%2 J • sodium borate (Na%O J s VRGLXPFKORULGH 1D&O J s &/5:TV P/ $GMXVWS+WR Glycine 0.01% and sucrose 5% pH 7.2 • glycine (CHNO J • sucrose (CHO J s &/5:TV P/ 6WHULOL]HE\ĺOWUDWLRQ Counter immunoelectrophoresis test Laboratory techniques in rabies Fifth edition Part 4. Demonstration of viral antibodies Penicillin solution 100 000 IU and streptomycin 200 mg/mL sSHQLFLOOLQ ,8 sVWUHSWRP\FLQ PJ 3UHYLRXVO\GLOXWHWKHSHQLFLOOLQWR,8P/DQGSUHSDUHVWUHSWRP\FLQZLWK WKLVVROXWLRQWRFRQWDLQJP/ 0LQHUDORLOPL[WXUH PDUFRODQGPRQWDQLGH sPDUFRO (VVR6WDQGDUG2LO&R P/ sPRQWDQLGH 6HSSLF&R3DULV)UDQFH P/ 6WHULOL]HWKHPLQHUDORLOPL[WXUHDW&IRUPLQ Counter immunoelectrophoresis test Laboratory techniques in rabies Fifth edition Appendix 1 List of contributors Jacques Barrat Maria Luiza Carrieri ANSES %XWDQWDQ,QVWLWXWH 0DO]ÆYLOOH)UDQFH 6ÀR3DXOR%UD]LO Helena B. C. Ruthne Batista Luciana Botelho Chaves Pasteur Institute Instituto Pasteur 6ÀR3DXOR%UD]LO 6ÀR3DXOR%UD]LO Emma M. Bentley Florence Cliquet University of Westminster ANSES London, United Kingdom 0DO]ÆYLOOH)UDQFH Jesse Blanton Laurent Dacheux Centers for Disease Control Pasteur Institute and Prevention 3DULV)UDQFH Atlanta, Georgia, USA Scott Dessain Juliano Bordignon /DQNHQDX,QVWLWXWHIRU Instituto Carlos Chagas 0HGLFDO5HVHDUFK )LRFUX]&XULWLED3DUDQ¾ Wynnewood, Pennsylvania, %UD]LO USA Leonard Both Elisa Eggerbauer 0HGLFLQHVDQG+HDOWKFDUH )ULHGULFK/RHIĻHU,QVWLWXW 'HYLFHV5HJXODWRU\$JHQF\ *UHLIVZDOG,QVHO5LHPV London, United Kingdom Germany Herve Bourhy Richard J. Ellis Pasteur Institute Animal and Plant Health 3DULV)UDQFH Agency Addlestone, Surrey, United Kingdom Graciane Maria Medeiros Caporale Instituto Pasteur 6ÀR3DXOR%UD]LO List of contributors Laboratory techniques in rabies Fifth edition Hildegund Ertl Cynthia Goldsmith The Wistar Institute Centers for Disease Control Philadelphia, Pennsylvania, and Prevention USA Atlanta, Georgia, USA Christine Fehlner-Gardiner F. Guedes &DQDGLDQ)RRG,QVSHFWLRQ Instituto Pasteur Agency 6ÀR3DXOR%UD]LO Ottawa, Canada Hans de Haard E.R. Fernandes Argenx Instituto Pasteur =ZLMQDDUGH%HOJLXP 6ÀR3DXOR%UD]LO Hajime Hatta Anthony R. Fooks .\RWR:RPHQpV8QLYHUVLW\ Animal and Plant Health .\RWR-DSDQ Agency Addlestone, Surrey, United Daniel J. Hicks Kingdom Animal and Plant Health Agency Richard Franka Addlestone, Surrey, United Centers for Disease Control Kingdom and Prevention Atlanta, Georgia, USA Anna Hultberg Argenx Conrad M. Freuling =ZLMQDDUGH%HOJLXP )ULHGULFK/RHIĻHU,QVWLWXW *UHLIVZDOG,QVHO5LHPV Satoshi Inoue Germany National Institute of Infectious Diseases Jinxin Gao 6KLQMXNX7RN\R-DSDQ Centers for Disease Control and Prevention Takuya Itou Atlanta, Georgia, USA 1LKRQ8QLYHUVLW\9HWHULQDU\ 5HVHDUFK&HQWHU Donna Gatewood .DQDJDZD-DSDQ Edge Consulting Ames, Iowa, USA Corinne Jallet Pasteur Institute of Guinea Florian Gohr &RQDNU\*XLQHD Aachen University Aachen, Germany Iana Suly Santos Katz Instituto Pasteur 6ÀR3DXOR%UD]LO List of contributors Laboratory techniques in rabies Fifth edition Ivanete Kotait Wilfred E. Marissen 9HQFR6D×GH$QLPDO Crucell Holland /RQGULQD3DUDQ¾%UD]LO Leiden, The Netherlands Ivan V. Kuzmin Wanda Markotter USDA University of Pretoria Ames, Iowa, USA Pretoria, South Africa Florence Larrous Denise A. Marston Pasteur Institute Animal and Plant Health 3DULV)UDQFH Agency Addlestone, Surrey, United Kingdom Monique Lechenne 8QLYHUVLW\RI%DVHO Lorraine M. McElhinney %DVHO6ZLW]HUODQG Animal and Plant Health Agency Yu Li Addlestone, Surrey, United Centers for Disease Control Kingdom and Prevention Atlanta, Georgia, USA Sharon Messenger California Department of Julian K.C. Ma Public Health 6W*HRUJHpV+RVSLWDO0HGLFDO 5LFKPRQG&DOLIRUQLD86$ School London, United Kingdom Susan Moore Kansas State University Anita Mahadevan 0DQKDWWDQ.DQVDV86$ 1DWLRQDO,QVWLWXWHRI0HQWDO Health and Neurosciences Wildeberg C. Moreira %DQJDORUH,QGLD Instituto Nacional de Controle GH4XDOLGDGHHP6D×GH Reeta Mani 5LRGH-DQHLUR%UD]LO 1DWLRQDO,QVWLWXWHRI0HQWDO Health and Neurosciences Wlamir C. Moura %DQJDORUH,QGLD Instituto Nacional de Controle GH4XDOLGDGHHP6D×GH Daria Llenaresas Manalo 5LRGH-DQHLUR%UD]LO 5HVHDUFK,QVWLWXWHIRU7URSLFDO 0HGLFLQH Thomas Müller 0XQWLQOXSD&LW\3KLOLSSLQHV )ULHGULFK/RHIĻHU,QVWLWXW *UHLIVZDOG,QVHO5LHPV Germany List of contributors Laboratory techniques in rabies Fifth edition Thirumeni Nagarajan Charles E. Rupprecht %LRORJLFDO(/LPLWHG LYSSA LLC Shameerpet, Hyderabad, Atlanta, Georgia, USA India Claude Sabeta Louis Nel $5&2QGHUVWHSRRUW University of Pretoria 9HWHULQDU\,QVWLWXWH Pretoria, South Africa Onderstepoort, South Africa Alejandro Núñez Markus Sack Animal and Plant Health Aachen University Agency Aachen, Germany Addlestone, Surrey, United Kingdom B. K. Chandrasekhar Sagar 1DWLRQDO,QVWLWXWHRI0HQWDO Lilian Orciari Health and Neurosciences Centers for Disease Control %HQJDOXUX,QGLD and Prevention Atlanta, Georgia, USA Michael Saunders Argenx Chun-Ho Park =ZLMQDDUGH%HOJLXP Kitasato University Higashi, Towada, Aomori, Jean Luc Schereffer -DSDQ ANSES 0DO]ÆYLOOH)UDQFH Evelyne Picard-Meyer ANSES Karin C. Scheffer 0DO]ÆYLOOH)UDQFH Instituto Pasteur 6ÀR3DXOR%UD]LO Luzia H. 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Teh Centers for Disease Control 6W*HRUJHpV+RVSLWDO0HGLFDO and Prevention School Atlanta, Georgia, USA London, United Kingdom Edward Wright Sanne Terryn University of Sussex Ablynx %ULJKWRQ8QLWHG.LQJGRP =ZLMQDDUGH%HOJLXP Mary Yager Noel Tordo Atlanta Health Associates, Pasteur Institute ,QF 3DULV)UDQFH Cumming, Georgia, USA Changchun Tu Pamela Yager $FDGHP\RI0LOLWDU\0HGLFDO Centers for Disease Control Sciences and Prevention Changchun, China Atlanta, Georgia, USA Steven Van Gucht Carlos Roberto Zanetti 6FLHQWLĺF,QVWLWXWHRI3XEOLF 8QLYHUVLGDGH)HGHUDOGH Health Santa Catarina %UXVVHOV%HOJLXP )ORULDQÐSROLV%UD]LO List of contributors Laboratory techniques in rabies Fifth edition Appendix 2 WHO collaborating centres on rabies, neurovirology, viral zoonoses and zoonoses control WHO Collaborating Centre for Reference and Research on Rabies, Institut 3DVWHXUUXHGX'RFWHXU5RX[3DULV)UDQFH +HDG'U+HUYÆ%RXUK\ HPDLO KHUYHERXUK\#SDVWHXUIU WHO Collaborating Centre on Research and Management on Zoonoses Control/DERUDWRU\RI5DELHVDQG:LOG$QLPDOVRI1DQF\$JULFXOWXUDODQG 9HWHULQDU\7HFKQRSROH32 0DO]ÆYLOOH)UDQFH +HDG'U)ORUHQFH&OLTXHW HPDLO ĻRUHQFHFOLTXHW#DQVHVIU WHO Collaborating Centre for the Characterization of Rabies and Rabies- related Viruses, Animal Health and Veterinary Laboratories Agency, Weybridge, 6XUUH\.71%8QLWHG.LQJGRP +HDG'U$QWKRQ\)RRNV HPDLO WRQ\IRRNV#DSKDJRYXN WHO Collaborating Centre for Rabies Surveillance and Research)ULHGULFK /RHIĻHU,QVWLWXW)HGHUDO5HVHDUFK,QVWLWXWHIRU$QLPDO+HDOWK6XGXIHU *UHLIVZDOG,QVHO5HLPV*HUPDQ\ +HDG'U7KRPDV0ÙOOHU HPDLO WKRPDVPXHOOHU#ĻLEXQGGH WHO Collaborating Centre for Traveller’s Health8QLYHUVLW\RI=XULFK +LUVFKHQJUDEHQ=XULFK6ZLW]HUODQG +HDG'U&KULVWRSK+DW] HPDLO FKULVWRSKKDW]#XQLEDVFK WHO Collaborating Centre for Control and Epidemiology of Rabies in Carnivores&DQDGLDQ)RRG,QVSHFWLRQ$JHQF\)DOORZĺHOG5RDG2WWDZD ON, Canada +HDG'U&KULVWLQH)HKOQHU*DUGLQHU HPDLO FKULVWLQHIHKOQHUJDUGLQHU#FDQDGDFD WHO Collaborating Centre for Neurovirology7KRPDV-HIIHUVRQ8QLYHUVLW\ 6RXWKWK6WUHHW3KLODGHOSKLD3$ 86$ +HDG'U0DWWKLDV6FKQHOO HPDLO PDWWKLDVVFKQHOO#MHIIHUVRQHGX WHO collaborating centers Laboratory techniques in rabies Fifth edition WHO Collaborating Centre for Reference and Research on Rabies, Wistar ,QVWLWXWH6SUXFH6WUHHW3KLODGHOSKLD3$ 86$ +HDG'U+LOGHJXQG(UWO HPDLO HUWO#ZLVWDUXSHQQHGX WHO Collaborating Centre for Reference and Research on Rabies, Centers for 'LVHDVH&RQWURODQG3UHYHQWLRQ&OLIWRQ5RDG$WODQWD*$86$ +HDG'U-HVVH%ODQWRQ HPDLO DVL#FGFJRY WHO Collaborating Centre in Rabies,QVWLWXW3DVWHXURI6ÀR3DXOR$Y3DXOLVWD 6ÀR3DXOR%UD]LO +HDG'U/XFLDQQD*RPHV'U-XOLDQD*DOHUD&DVWLOKR HPDLOSDVWHXU#SDVWHXU VDXGHVSJRYEUMJFDVWLOKR#SDVWHXUVDXGHVSJRYEU WHO Collaborating Centre for Rabies Epidemiology'LYLVLRQRI=RRQRVLV 1DWLRQDO&HQWUHIRU'LVHDVH&RQWURO6KDP1DWK'HOKL,QGLD +HDG'U0DOD&KDEUD HPDLO PDODFKKDEUD#\DKRRFRLQ WHO Collaborating Centre for Reference and Research in Rabies, Department RI1HXURYLURORJ\1DWLRQDO,QVWLWXWHRI0HQWDO+HDOWKDQG1HXURVFLHQFHV32%R[ %DQJDORUH,QGLD +HDG3URIHVVRU95DYL'U5HHWD0DQL HPDLO YLUXVUDYL#JPDLOFRPGUUHHWD# JPDLOFRP WHO Collaborating Centre for Research on Rabies Pathogenesis and Prevention4XHHQ6DRYDEKD0HPRULDO,QVWLWXWH7KDL5HG&URVV6RFLHW\ 5DPD,95RDG%DQJNRN7KDLODQG +HDG3URIHVVRU9LVLWK6LWSULMD HPDLO VLWSULMD#\DKRRFRP DQG'U3DNPDQHH 1DUXPRO HPDLO QSDNPDQHH#\DKRRFRP WHO Collaborating Centre for Research and Training on Viral Zoonoses, 'HSDUWPHQWRI0HGLFLQH1HXURYLURORJ\'LYLVLRQ)DFXOW\RI0HGLFLQH &KXODORQJNRUQ8QLYHUVLW\5DPD,95RDG%DQJNRN7KDLODQG +HDG3URIHVVRU7KLUDYDW+HPDFKXGKD HPDLO IPHGWKP#JPDLOFRP DQG'U 6XSDSRUQ:DFKDUDSOXHVDGHH HPDLO VSZD#KRWPDLOFRP WHO collaborating centers ISBN: 978-92-4-151515-3